Methods for assessing cell surface glycosylation

ABSTRACT

Provided herein are methods for assessing cell surface glycans, e.g., N-glycans, by assessing a sample of released surface glycans, and determining the presence, absence, or level of glycans present in the sample. Also provided are methods of assaying and/or evaluating a cell composition by assessing the cell surface glycan profile of the cell composition and comparing the profile to a reference sample. Methods for manufacturing and/or culturing a plurality of cell compositions having consistent surface glycan expression with low variability are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

The application is a National Stage application under 35 U.S.C. § 371 ofInternational Application No. PCT/US2018/027666, filed on Apr. 13, 2018,which claims the benefit of priority to U.S. provisional patentapplications 62/485,897, filed Apr. 14, 2017, entitled “METHODS FORASSESSING CELL SURFACE GLYCOSYLATION” and U.S. provisional applicationNo. 62/515,515, filed Jun. 5, 2017, entitled “METHODS FOR ASSESSING CELLSURFACE GLYCOSYLATION,” the contents of each of which are incorporatedby reference in their entirety.

INCORPORATION BY REFERENCE OF SEQUENCE LISTING

The present application is being filed along with a Sequence Listing inelectronic format. The Sequence Listing is provided as a file entitled735042010800SeqList.TXT, created Oct. 10, 2019, which is 37,300 bytes insize. The information in the electronic format of the Sequence Listingis incorporated by reference in its entirety.

FIELD

Provided herein are methods for assessing cell surface glycans, e.g.,N-glycans, by assessing a sample of released surface glycans, anddetermining the presence, absence, or level of glycans present in thesample. Also provided are methods of assaying and/or evaluating a cellcomposition by assessing the cell surface glycan profile of the cellcomposition and comparing the profile to a reference sample. Methods formanufacturing and/or culturing a plurality of cell compositions havingconsistent surface glycan expression with low variability are alsoprovided.

BACKGROUND

Glycans are among the principal components of a cell. Nearly all humanmembrane proteins, as well as numerous intracellular proteins, are co-and post-translationally modified by the covalent addition of glycans.In particular, N-glycans are post-translational modifications toproteins that can have far reaching impact to structure and function. Ata cellular level, glycosylation has been implicated in cell signaling,adhesion, homing properties and other functional activities. Thereexists a need in the art for additional methods to measure and identifyglycans, e.g., N-glycans, that are expressed on the surface of cells.

SUMMARY

Provided herein are methods for assessing cell surface glycans, themethods comprising: (a) incubating a test composition comprising aplurality of cells under conditions to release one or more glycans fromthe surface of cells in the test composition, wherein a samplecomprising one or more cell surface glycans is generated; and (b)determining the presence, absence, identity and/or level of glycanspresent in the sample, thereby assessing the cell surface glycan profileof the sample.

Also provided herein are methods for assessing cell surface glycans, themethods comprising determining the presence, absence, identity and/orlevel of glycans present in a sample, thereby assessing the cell surfaceglycan profile of the sample, wherein the sample comprises one or moreglycans released from the surface of cells present in a test compositioncomprising a plurality of cells after incubation of the test compositionunder conditions to release the one or more glycans.

In some embodiments of any of the provided methods, the glycans areN-glycans. In particular embodiments of any of the provided methods,cells in the test cell composition comprise whole or intact cells. Insome embodiments of any of the provided methods, the cells are livecells. In certain embodiments of any of the provided methods, the testcell composition is not homogenized or sonicated prior to theincubation; and/or the test cell composition is not incubated with aprotease prior to the incubation, optionally wherein the protease istrypsin; and/or the cells in the test cell composition, prior to orduring the incubation, are not contacted with an agent to extract one ormore cell surface or membrane proteins, optionally wherein the agent isa detergent or protease, optionally trypsin; and/or less than 10% of thecells are lysed and/or ruptured during the incubation.

In particular embodiments of any of the provided methods, the test cellcomposition comprises no more than 5×10⁶ cells. In some embodiments ofany of the provided methods, the test cell composition comprises between1×10⁶ cells and 5×10⁶ cells, inclusive. In certain embodiments of any ofthe provided methods, the test cell composition comprises aconcentration of no more than 1×10⁸ cells/mL. In particular embodimentsof any of the provided methods, the test cell composition comprises aconcentration of between 1×10⁵ cells/mL and 1×10⁸ cells/mL, inclusive,between 1×10⁶ cells/mL and 5×10⁷ cells/mL, inclusive, or between 5×10⁶cells/mL and 2.5×10⁷ cells/mL, inclusive. In some embodiments of any ofthe provided methods, the incubation is carried out in the presence ofan N-glycosidase. In certain embodiments of any of the provided methods,the N-glycosidase is a peptide N-glycosidase (PNGase) F. In particularembodiments of any of the provided methods, the PNGase F is recombinant.

In some embodiments of any of the provided methods, the one or moreglycans are one or more N-glycans, and wherein the method comprises: (i)incubating between 1×10⁶ and 5×10⁶ cells from the test composition witha recombinant PNGase F under conditions to release the one or moreN-glycans from the surface of the cells of the test composition; (ii)labeling the one or more N-glycans with a detectable label, optionally afluorescent label; and (iii) determining the presence, absence, or levelof the labeled N-glycans, thereby assessing the cell surface glycanprofile of the sample.

In certain embodiments of any of the provided methods, the test cellcomposition comprises about 1×10⁶ to 2.5×10⁶ cells. In particularembodiments of any of the provided methods, the test cell compositioncomprises a concentration of between 1×10⁶ cells/mL and 5×10⁷ cells/mL.In some embodiments of any of the provided methods, the PNGase Fcomprises a PGNase F of Flavobacterium meningosepticum, or a portion ormutant thereof that is enzymatically active. In certain embodiments ofany of the provided methods, the PNGase F comprises the amino acidsequence set forth in SEQ ID NO: 1 or a portion or mutant thereof thatis enzymatically active, or an amino acid sequence that exhibits atleast 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99% or more sequence identity to SEQ ID NO: 1 or is a portionthereof that is enzymatically active. In particular embodiments of anyof the provided methods, the PNGase F comprises the amino acid sequenceset forth in SEQ ID NO: 1. In some embodiments of any of the providedmethods, the PNGase F comprises a tag, optionally an affinity tag. Incertain embodiments of any of the provided methods, the tag is apoly-histidine (His-tag).

In particular embodiments of any of the provided methods, the PNGase Fis greater than or greater than about 90%, greater than or greater thanabout 92%, greater than or greater than about 95%, or greater than orgreater than about 98% pure; and/or the PNGase F comprises less than orless than about 10%, less than or less than about 8%, less than or lessthan about 5%, less than or less than about 2% non-PNGase F proteincontaminants; and/or the PNGase F is greater than or greater than about90%, greater than or greater than about 92%, greater than or greaterthan about 95%, or greater than or greater than about 98% homogeneous,optionally as determined by SDS-PAGE and protein staining, optionallyCoomasie Blue staining.

In some embodiments of any of the provided methods, the N-glycosydase,optionally PNGase F, is in an enzymatically effective amount to releasethe one or more N-glycans from a native or non-denatured glycoprotein orglycoproteins and/or from the cells of the cell composition afterincubation for no more than 12 hours at a temperature between 35° C. and39° C., optionally about 37° C. In certain embodiments of any of theprovided methods, the enzymatically effective amount is an amount torelease the one or more N-glycans after incubation for no more than 15minutes to 3 hours or 30 minutes to 2 hours, at a temperature between25° C. and 39° C. or between 35° C. and 39° C., each inclusive,optionally about 37° C. In particular embodiments of any of the providedmethods, the enzymatically effective amount of PNGase F releases greaterthan 50%, greater than 55%, greater than 60%, greater than 65%, greaterthan 70%, greater than 75%, greater than 80%, greater than 85%, greaterthan 90%, greater than 95%, greater than 99% of N-glycans present on theglycoprotein or glycoproteins and/or present on the surface of the cellcomposition. In some embodiments of any of the provided methods, theconditions of the incubation are sufficient to effect release of greaterthan 50%, greater than 55%, greater than 60%, greater than 65%, greaterthan 70%, greater than 75%, greater than 80%, greater than 85%, greaterthan 90%, greater than 95%, greater than 99% N-glycans present on thesurface of the test cell composition.

In certain embodiments of any of the provided methods, the amount ofN-glycosidase, optionally PNGase F, is 1 unit to 5000 units, 1 unit to1000 units, 1 unit to 500 units, 1 unit to 250 units, 1 unit to 100units, 1 unit to 50 units, 1 unit to 25 units, 25 units to 5000 units,25 units to 1000 units, 25 units to 500 units, 25 units to 250 units, 25units to 100 units, 25 units to 50 units, 50 units to 5000 units, 50units to 1000 units, 50 units to 500 units, 50 units to 250 units, 50units to 100 units, 100 units to 5000 units, 100 units to 1000 units,100 units to 500 units, 100 units to 250 units, 250 units to 5000 units,250 units to 1000 units, 250 units to 500 units, 500 units to 5000units, 500 units to 1000 units, or 1000 units to 5000 units, eachinclusive. In particular embodiments of any of the provided methods, theamount of N-glycosidase, optionally PNGase F, is greater than or greaterthan about or is or is about 1 unit, 5 units, 10 units, 15 units, 20units, 25 units, 50 units, 100 units, 250 units, 500 units, 1000 units,2500 units or 5000 units. The method of claim 27 or claim 28, whereinone unit is an amount of the N-glycosidase, optionally PNGase F,sufficient to catalyze the deglycosylation of 1 nanomole of denaturedRibonuclease B (RNase B) in 30 minutes at 37° C. In some embodiments ofany of the provided methods, 500 units is an amount of theN-glycosidase, optionally PNGase F, sufficient to catalyze thedeglycosylation of 10 μg of Ribonuclease B (RNase B) incubated in 1XPBSfor 5-10 minutes at 37° C. or room temperature.

In certain embodiments of any of the provided methods, the incubatingthe test composition is for an amount of time that between or betweenabout 5 minutes and 12 hours, 30 minutes and 6 hours or 1 hour and 3hours, each inclusive. In particular embodiments of any of the providedmethods, the incubating the test composition is for at least or at leastabout or is or is about 5 minutes, about 10 minutes, about 15 minutes,30 minutes, 1 hour, 2 hours 3 hours, 4 hours, 5 hours or 6 hours. Insome embodiments of any of the provided methods, the incubating the testcomposition is for about 30 minutes. In certain embodiments of any ofthe provided methods, the incubating the test composition is at atemperature between 25° C. and 39° C. or between 35° C. and 39° C. Inparticular embodiments of any of the provided methods, the incubatingthe test composition is at a temperature of about 37° C. In someembodiments of any of the provided methods, the incubating the testcomposition is for about 30 minutes at a temperature of about 37° C.

In certain embodiments of any of the provided methods, prior to thedetermining the presence, absence, identity and/or level of glycanspresent in a sample, the method further comprises labeling glycans fromthe sample with a detectable label, optionally a fluorescent label. Inparticular embodiments of any of the provided methods, the label is afluorescent label and the fluorescent label is or comprises2-aminobenzamide (2-AB), 2-aminobenzoic acid (2-AA), 2-aminopyridine(PA), 2-Aminoacridone (AMAC), 2-aminonaphthalene trisulfonic acid(ANTS), and 1-aminopyrene-3,6,8-trisulfonic acid (APTS),3-(Acetylamino)-6-aminoacridin (AA-Ac), 6-Aminoquinoline (6-AQ),7-Aminomethyl-coumarin (AMC), 2-Amino (6-amido-biotinyl) pyridine (BAP),9-Fluorenylmethoxycarbonyl (FMOC)-hydrazide,1,2-Diamino-4,5-methylenedioxy-benzene (DMB), or o-Phenylenediamine(OPD). In some embodiments of any of the provided methods, thefluorescent label comprises a quinolinyl fluorophore. In certainembodiments of any of the provided methods, the fluorescent labelcomprises a carbamate tagging group. In particular embodiments of any ofthe provided methods, the fluorescent label comprises a basic tertiaryamine. In some embodiments of any of the provided methods, thefluorescent label comprises a carbamate tagging group, a quinolonefluorophore, and a tertiary amine.

In certain embodiments of any of the provided methods, prior todetermining the presence, absence, identity and/or level of the one ormore glycans, the sample is subjected to glycan purification orenrichment. In particular embodiments of any of the provided methods,glycan purification or enrichment is carried out by solid phaseextraction (SPE).

In some embodiments of any of the provided methods, determining thepresence, absence, or level of the one or more glycans comprisessubjecting the sample to mass spectrometry. In certain embodiments ofany of the provided methods, the mass spectrometry is electrosprayionization mass spectrometry (ESI-MS), turbospray ionization massspectrometry, nanospray ionization mass spectrometry, thermosprayionization mass spectrometry, sonic spray ionization mass spectrometry,surface enhanced laser desorption ionization mass spectrometry(SELDI-MS) and matrix assisted laser desorption/ionization massspectrometry (MALDI-MS). In particular embodiments of any of theprovided methods, the mass spectrometry is MALDI-MS.

In some embodiments of any of the provided methods, determining thepresence, absence, or level of glycans comprises subjecting the sampleto liquid chromatography (LC) followed by mass spectrometry. In certainembodiments of any of the provided methods, the liquid chromatography ishigh performance liquid chromatography (HPLC), ultra high performanceliquid chromatography (UHPLC), or ultra performance liquidchromatography (UPLC). In particular embodiments of any of the providedmethods, the liquid chromatography is ultra performance liquidchromatography (UPLC). In some embodiments of any of the providedmethods, the liquid chromatography and mass spectrometry are carried outonline. In certain embodiments of any of the provided methods, theliquid chromatography is selected from normal phase (NP-), reverse phase(RP) and hydrophilic interaction chromatography (HILIC). In particularembodiments of any of the provided methods, the liquid chromatography ishydrophilic interaction chromatography (HILIC).

In some embodiments of any of the provided methods, the massspectrometry comprises electrospray ionization mass spectrometry(ESI-MS), turbospray ionization mass spectrometry, nanospray ionizationmass spectrometry, thermospray ionization mass spectrometry or sonicspray ionization mass spectrometry. In some embodiments of any of theprovided methods, the mass spectrometry comprises ESI-MS. In particularembodiments of any of the provided methods, the mass spectrometrycomprises tandem mass spectrometry (MS/MS). In certain embodiments ofany of the provided methods, the mass spectrometry comprises tandem ESImass spectrometry (ESI-MS/MS).

In certain embodiments of any of the provided methods, the massspectrometer that performs the mass spectrometry comprises one or moreof a quadrupole, ion trap, time of flight (TOF), or Fourier transformion cyclotron resonance mass analyzer. In particular embodiments of anyof the provided methods, the mass spectrometer comprises an ion trapmass analyzer that is a three-dimensional quadrupole ion trap, acylindrical ion trap, a linear quadrupole ion trap, or an Orbitrap massanalyzer. In some embodiments of any of the provided methods, the massspectrometer is a quadrupole-Orbitrap mass spectrometer.

In certain embodiments of any of the provided methods, the determiningthe presence, absence, identity and/or level of the one or more glycanscomprises analyzing one or more glycan structure or structures forbranching, linkages between monosaccharides and/or location ofmonosaccharides. In particular embodiments of any of the providedmethods, the one or more glycans comprises high mannose N-glycans,bisected and Sialyl Lewisx N-glycans, and/or N-acetyl lactosaminecontaining N-glycans. In some embodiments of any of the providedmethods, the one or more glycans comprises a fucosylated biantennarycomplex glycan having no reducing end terminal galactose residues, afucosylated biantennary complex glycan having one reducing end terminalgalactose residue, a fucosylated biantennary complex glycan having tworeducing end terminal galactose residues, a biantennary complex glycanhaving no reducing end terminal galactose residues, a biantennarycomplex glycan having one reducing end terminal galactose residue, abiantennary complex glycan having two reducing end terminal galactoseresidues, a fucosylated biantennary complex glycan having two galactoseresidues and one N-acetylneuraminic acid residue, a fucosylatedbiantennary complex glycan having two galactose residues and twoN-acetylneuraminic acid residues, a biantennary complex glycan havingtwo galactose residues and two N-acetylneuraminic acid residues, a highmannose glycan having five mannose residues, a high mannose glycanhaving six mannose residues, a high mannose glycan having seven mannoseresidues, a high mannose glycan having eight mannose residues, and/or ahigh mannose glycan having nine mannose residues.

In certain embodiments of any of the provided methods, the determiningthe presence, absence, or level of glycans present in the samplecomprises determining the presence, absence, or level of at least 25, atleast 50, at least 60, at least 70, at least 80, at least 90, at least100, or at least 200 different species of glycans. In particularembodiments of any of the provided methods, the presence or level of aglycan species present in the sample is determined if at least 100 amol,at least 500 amol, at least 1 fmol, at least 5 fmol, or at least 10 fmolof the glycan species is present in the sample. In some embodiments ofany of the provided methods, the presence or level of a glycan speciespresent in the sample is determined if glycan species makes up at least0.00001%, 0.00005%, 0.0001%, 0.0005%, 0.001%, 0.005%, or 0.01% of thetotal glycans in the sample. In certain embodiments of any of theprovided methods, the species of glycans are species of N-glycans.

In particular embodiments of any of the provided methods, the test cellcomposition is or comprises at least a portion of a source cellcomposition comprising the plurality of cells. In some embodiments ofany of the provided methods, the cells comprise mammalian cells or thetest cell composition comprises mammalian cells. In certain embodimentsof any of the provided methods, the cells comprise human cells or thetest cell composition comprises human cells. In particular embodimentsof any of the provided methods, the cells comprise stem cells or thetest cell composition comprises stem cells. In some embodiments of anyof the provided methods, the stem cell is an induced pluripotent stemcell (iPSC). In certain embodiments of any of the provided methods, thetest cell composition comprises cells present in an apheresis product ora leukapheresis product or cells derived therefrom. In particularembodiments of any of the provided methods, the cells comprise immunecells, white blood cells, peripheral blood mononuclear cells (PBMC),lymphocytes, or unfractionated T cells; or the test cell compositioncomprises immune cells, white blood cells, peripheral blood mononuclearcells (PBMC), lymphocytes, or unfractionated T cells. In someembodiments of any of the provided methods, the cells comprise an immunecell or the test cell composition comprises immune cells. In certainembodiments of any of the provided methods, the immune cell is a T cell,B cell, macrophage, neutrophil, natural killer (NK) cell or dendriticcell. In particular embodiments of any of the provided methods, thecells comprise T cells that are CD4+ and/or CD8+ T cells or the testcell composition comprises T cells that are CD4+ and/or CD8+ T cells.

In some embodiments of any of the provided methods, the test cellcomposition comprises: cells isolated from a biological sample byimmunoaffinity-based methods; and/or cells transduced with a viralvector encoding a recombinant protein; and/or cell incubated in thepresence of one or more test agents, optionally one more peptide,protein, polypeptide, nucleic acid, small molecule; and/or cellsactivated and/or expanded in the presence of one or more stimulatingconditions; and/or cryopreserved cells and/or cells comprising acryoprotectant; and/or cells formulated for administration to a subject,optionally in the presence of a pharmaceutically acceptable excipient.

In certain embodiments of any of the provided methods, the test agent isa candidate for modulating the growth, proliferation, viability,differentiation, intracellular signaling, activation and/or expansion ofone or more cells in the test cell composition. In particularembodiments of any of the provided methods, the stimulating conditioncomprises incubation with a stimulatory reagent capable of activatingone or more intracellular signaling domains of one or more components ofa TCR complex and/or one or more intracellular signaling domains of oneor more costimulatory molecules. In some embodiments of any of theprovided methods, the stimulatory reagent comprises a primary agent thatspecifically binds to a member of a TCR complex and a secondary agentthat specifically binds to a T cell costimulatory molecule. In certainembodiments of any of the provided methods, the primary agentspecifically binds to CD3 and/or the costimulatory molecule is selectedfrom the group consisting of CD28, CD137 (4-1-BB), OX40, or ICOS. Inparticular embodiments of any of the provided methods, stimulatoryreagent comprises an anti-CD3 antibody or antigen binding fragmentthereof and an anti-CD28 antibody or an antigen-binding fragmentthereto. In some embodiments of any of the provided methods, the primaryand secondary agents comprise antibodies and/or are present on thesurface of a solid support. In certain embodiments of any of theprovided methods, the solid support is or comprises a bead.

In particular embodiments of any of the provided methods, the cellsexpress a recombinant receptor or the test composition comprises cellsexpressing a recombinant receptor. In certain embodiments of any of theprovided methods, the recombinant receptor is or comprises a chimericreceptor and/or a recombinant antigen receptor. In some embodiments ofany of the provided methods, the recombinant receptor is capable ofbinding to a target antigen that is associated with, specific to, and/orexpressed on a cell or tissue of a disease, disorder or condition. Inparticular embodiments of any of the provided methods, the disease,disorder or condition is an infectious disease or disorder, anautoimmune disease, an inflammatory disease, or a tumor or a cancer. Incertain embodiments of any of the provided methods, the target antigenis a tumor antigen.

In some embodiments of any of the provided methods, the target antigenis selected from among ROR1, B cell maturation antigen (BCMA), carbonicanhydrase 9 (CAIX), tEGFR, Her2/neu (receptor tyrosine kinase erbB2),L1-CAM, CD19, CD20, CD22, mesothelin, CEA, and hepatitis B surfaceantigen, anti-folate receptor, CD23, CD24, CD30, CD33, CD38, CD44, EGFR,epithelial glycoprotein 2 (EPG-2), epithelial glycoprotein 40 (EPG-40),EPHa2, erb-B2, erb-B3, erb-B4, erbB dimers, EGFR vIII, folate bindingprotein (FBP), FCRLS, FCRHS, fetal acetylcholine receptor, GD2, GD3,HMW-MAA, IL-22R-alpha, IL-13R-alpha2, kinase insert domain receptor(kdr), kappa light chain, Lewis Y, L1-cell adhesion molecule, (L1-CAM),Melanoma-associated antigen (MAGE)-A1, MAGE-A3, MAGE-A6, Preferentiallyexpressed antigen of melanoma (PRAME), survivin, TAG72, B7-H6, IL-13receptor alpha 2 (IL-13Rα2), CA9, GD3, HMW-MAA, CD171, G250/CAIX, HLA-AIMAGE A1, HLA-A2 NY-ESO-1, PSCA, folate receptor-a, CD44v6, CD44v7/8,avb6 integrin, 8H9, NCAM, VEGF receptors, 5T4, Foetal AchR, NKG2Dligands, CD44v6, dual antigen, a cancer-testes antigen, mesothelin,murine CMV, mucin 1 (MUC1), MUC16, PSCA, NKG2D, NY-ESO-1, MART-1, gp100,oncofetal antigen, ROR1, TAG72, VEGF-R2, carcinoembryonic antigen (CEA),Her2/neu, estrogen receptor, progesterone receptor, ephrinB2, CD123,c-Met, GD-2, O-acetylated GD2 (OGD2), CE7, Wilms Tumor 1 (WT-1), acyclin, cyclin A2, CCL-1, CD138, a pathogen-specific antigen and anantigen associated with a universal tag. In particular embodiments ofany of the provided methods, the recombinant receptor is or comprises afunctional non-TCR antigen receptor or a TCR or antigen-binding fragmentthereof. In certain embodiments of any of the provided methods, therecombinant receptor is a chimeric antigen receptor (CAR).

In certain embodiments, the target antigen is selected from among αvβ6integrin (avb6 integrin), B cell maturation antigen (BCMA), B7-H3,B7-H6, carbonic anhydrase 9 (CA9, also known as CAIX or G250), acancer-testis antigen, cancer/testis antigen 1B (CTAG, also known asNY-ESO-1 and LAGE-2), carcinoembryonic antigen (CEA), a cyclin, cyclinA2, C-C Motif Chemokine Ligand 1 (CCL-1), CD19, CD20, CD22, CD23, CD24,CD30, CD33, CD38, CD44, CD44v6, CD44v7/8, CD123, CD133, CD138, CD171,chondroitin sulfate proteoglycan 4 (CSPG4), epidermal growth factorprotein (EGFR), truncated epidermal growth factor protein (tEGFR), typeIII epidermal growth factor receptor mutation (EGFR vIII), epithelialglycoprotein 2 (EPG-2), epithelial glycoprotein 40 (EPG-40), ephrinB2,ephrine receptor A2 (EPHa2), estrogen receptor, Fc receptor like 5(FCRLS; also known as Fc receptor homolog 5 or FCRHS), fetalacetylcholine receptor (fetal AchR), a folate binding protein (FBP),folate receptor alpha, ganglioside GD2, O-acetylated GD2 (OGD2),ganglioside GD3, glycoprotein 100 (gp100), glypican-3 (GPC3), G ProteinCoupled Receptor 5D (GPCRSD), Her2/neu (receptor tyrosine kinaseerb-B2), Her3 (erb-B3), Her4 (erb-B4), erbB dimers, Human high molecularweight-melanoma-associated antigen (HMW-MAA), hepatitis B surfaceantigen, Human leukocyte antigen A1 (HLA-A1), Human leukocyte antigen A2(HLA-A2), IL-22 receptor alpha(IL-22Rα), IL-13 receptor alpha 2(IL-13Rα2), kinase insert domain receptor (kdr), kappa light chain, L1cell adhesion molecule (L1-CAM), CE7 epitope of L1-CAM, Leucine RichRepeat Containing 8 Family Member A (LRRC8A), Lewis Y,Melanoma-associated antigen (MAGE)-A1, MAGE-A3, MAGE-A6, MAGE-A10,mesothelin (MSLN), c-Met, murine cytomegalovirus (CMV), mucin 1 (MUC1),MUC16, natural killer group 2 member D (NKG2D) ligands, melan A(MART-1), neural cell adhesion molecule (NCAM), oncofetal antigen,Preferentially expressed antigen of melanoma (PRAME), progesteronereceptor, a prostate specific antigen, prostate stem cell antigen(PSCA), prostate specific membrane antigen (PSMA), Receptor TyrosineKinase Like Orphan Receptor 1 (ROR1), survivin, Trophoblast glycoprotein(TPBG also known as 5T4), tumor-associated glycoprotein 72 (TAG72),Tyrosinase related protein 1 (TRP1, also known as TYRP1 or gp75),Tyrosinase related protein 2 (TRP2, also known as dopachrometautomerase, dopachrome delta-isomerase or DCT), vascular endothelialgrowth factor receptor (VEGFR), vascular endothelial growth factorreceptor 2 (VEGFR2), Wilms Tumor 1 (WT-1), a pathogen-specific orpathogen-expressed antigen, or an antigen associated with a universaltag, and/or biotinylated molecules, and/or molecules expressed by HIV,HCV, HBV or other pathogens.

Provided herein are methods of assaying a cell composition, the methodscomprising: (a) assessing the cell surface glycan profile in a samplefrom a test cell composition comprising a plurality of cells accordingto the methods provided herein; and (b) comparing the cell surfaceglycan profile of the sample to the cell surface glycan profile of areference sample. Provided herein are methods of assaying a cellcomposition, the methods comprising comparing the cell surface glycanprofile of a sample compared to the cell surface profile of a referencesample, wherein cell surface glycan profile of the sample is or has beendetermined according to the method of any of the methods provided hereinfrom a test cell composition comprising a plurality of cells.

In certain embodiments of any of the provided methods, the cell surfaceglycan profile comprises at least 25, at least 50, at least 60, at least70, at least 80, at least 90, at least 100, or at least 200 differentspecies of glycans, optionally different species of N-glycans. Inparticular embodiments of any of the provided methods, the cell surfaceglycan profile comprises high mannose N-glycans, bisected and SialylLewis^(X) N-glycans, and/or N-acetyl lactosamine containing N-glycans.In some embodiments of any of the provided methods, the cell surfaceglycan profile comprise a fucosylated biantennary complex glycan havingno reducing end terminal galactose residues, a fucosylated biantennarycomplex glycan having one reducing end terminal galactose residue, afucosylated biantennary complex glycan having two reducing end terminalgalactose residues, a biantennary complex glycan having no reducing endterminal galactose residues, a biantennary complex glycan having onereducing end terminal galactose residue, a biantennary complex glycanhaving two reducing end terminal galactose residues, a fucosylatedbiantennary complex glycan having two galactose residues and oneN-acetylneuraminic acid residue, a fucosylated biantennary complexglycan having two galactose residues and two N-acetylneuraminic acidresidues, a biantennary complex glycan having two galactose residues andtwo N-acetylneuraminic acid residues, a high mannose glycan having fivemannose residues, a high mannose glycan having six mannose residues, ahigh mannose glycan having seven mannose residues, a high mannose glycanhaving eight mannose residues, and/or a high mannose glycan having ninemannose residues.

In certain embodiments of any of the provided methods, the cell surfaceglycan profile comprises the glycans in Table E1 or a subset thereof. Inparticular embodiments of any of the provided methods, the referencesample is a reference standard comprising a release specification, alabel requirement or a compendia specification. In some embodiments ofany of the provided methods, the cell composition is released fortreatment of a subject only if the cell surface glycan profile of thecomposition is substantially the same as the reference sample and/or ifthe percent of a target glycan or each of a plurality of target glycansto the total glycans present in the sample differs by no more than 25%,no more than 20% or no more than 10% from the percent of the targetglycan or each of the plurality of target glycans to the total glycanspresent in the reference sample. In certain embodiments of any of theprovided methods, the target glycans comprise at least 25, at least 50,at least 60, at least 70, at least 80, at least 90, at least 100, or atleast 200 different species of glycans.

In particular embodiments of any of the provided methods, the targetglycan or glycans comprise high mannose N-glycans, bisected and SialylLewis^(X) N-glycans, and/or N-acetyl lactosamine containing N-glycans.In some embodiments of any of the provided methods, the target glycan orglycans comprise a fucosylated biantennary complex glycan having noreducing end terminal galactose residues, a fucosylated biantennarycomplex glycan having one reducing end terminal galactose residue, afucosylated biantennary complex glycan having two reducing end terminalgalactose residues, a biantennary complex glycan having no reducing endterminal galactose residues, a biantennary complex glycan having onereducing end terminal galactose residue, a biantennary complex glycanhaving two reducing end terminal galactose residues, a fucosylatedbiantennary complex glycan having two galactose residues and oneN-acetylneuraminic acid residue, a fucosylated biantennary complexglycan having two galactose residues and two N-acetylneuraminic acidresidues, a biantennary complex glycan having two galactose residues andtwo N-acetylneuraminic acid residues, a high mannose glycan having fivemannose residues, a high mannose glycan having six mannose residues, ahigh mannose glycan having seven mannose residues, a high mannose glycanhaving eight mannose residues, and/or a high mannose glycan having ninemannose residues. In certain embodiments of any of the provided methods,the target glycan or glycans comprise the glycans present in Table E1 ora subset thereof. In particular embodiments of any of the providedmethods, the target glycan or glycans comprise the glycans detectable inthe cell surface glycan profile. In some embodiments of any of theprovided methods, the reference sample is a cell surface glycan profilefrom a different cell composition.

In certain embodiments of any of the provided methods, the differentcell composition is from a different stage of a manufacturing processfor producing the test cell composition or a source cell compositionfrom which the test composition has been derived or obtained, whereinthe stage of the manufacturing process optionally is a prior stage ofthe manufacturing process. In particular embodiments of any of theprovided methods, the manufacturing process comprises one or more stagesselected from: cells isolated from a biological sample by leukapheresisor apheresis; cells selected from a biological sample byimmunoaffinity-based methods; and/or cells introduced with a recombinantnucleic acid, optionally a viral vector encoding a recombinant protein;and/or cell incubated in the presence of one or more test agents,optionally one more peptide, protein, polypeptide, nucleic acid, smallmolecule; and/or cells activated and/or expanded in the presence of oneor more stimulating conditions; and/or cryopreservation of cells in thepresence of a cryoprotectant; and/or cells formulated for administrationto a subject, optionally in the presence of a pharmaceuticallyacceptable excipient.

In some embodiments of any of the provided methods, a difference in theglycan profile between the test composition and reference sampleindicates one or more differences is present in the cells among thecells produced at the different stages in the manufacturing process. Incertain embodiments of any of the provided methods, the difference inthe glycan profile exists if the cell surface glycan profile of thecomposition is substantially different from the reference sample and/orif the percent of a target glycan or each of the one or more targetglycans to the total glycans present in the sample differs by greaterthan or greater than about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%or more from the percent of the target glycan or each of the one or moretarget glycans to the total glycans present in the reference sample. Inparticular embodiments of any of the provided methods, the targetglycans comprise at least 25, at least 50, at least 60, at least 70, atleast 80, at least 90, at least 100, or at least 200 different speciesof glycans, optionally wherein the species of glycans are N-glycans.

In some embodiments of any of the provided methods, the target glycan orglycans comprise high mannose N-glycans, bisected and Sialyl Lewis'N-glycans, and/or N-acetyl lactosamine containing N-glycans. In certainembodiments of any of the provided methods, the target glycan or glycanscomprise a fucosylated biantennary complex glycan having no reducing endterminal galactose residues, a fucosylated biantennary complex glycanhaving one reducing end terminal galactose residue, a fucosylatedbiantennary complex glycan having two reducing end terminal galactoseresidues, a biantennary complex glycan having no reducing end terminalgalactose residues, a biantennary complex glycan having one reducing endterminal galactose residue, a biantennary complex glycan having tworeducing end terminal galactose residues, a fucosylated biantennarycomplex glycan having two galactose residues and one N-acetylneuraminicacid residue, a fucosylated biantennary complex glycan having twogalactose residues and two N-acetylneuraminic acid residues, abiantennary complex glycan having two galactose residues and twoN-acetylneuraminic acid residues, a high mannose glycan having fivemannose residues, a high mannose glycan having six mannose residues, ahigh mannose glycan having seven mannose residues, a high mannose glycanhaving eight mannose residues, and/or a high mannose glycan having ninemannose residues. In particular embodiments of any of the providedmethods, the target glycan or glycans comprise the glycans present inTable E1 or a subset thereof. In some embodiments of any of the providedmethods, the target glycan or glycans comprise the glycans detectable inthe cell surface glycan profile.

In certain embodiments of any of the provided methods, the one moredifferences is associated with a functional activity or phenotype of thecells. In particular embodiments of any of the provided methods, thefunctional activity or phenotype comprises one or more of masking of acell surface marker, a metabolic activity, differentiation state,proliferative or expansion capacity, activation state, cytolyticactivity, signaling activity, an adhesion property, or a homingproperty. Some embodiments of any of the provided methods furthercomprise modulating or changing the process for manufacturing the cellcomposition. In certain embodiments of any of the provided methods, thereference standard comprises an average or median of the presence,absence, identity and/or level of the one or more target glycan orglycans among a plurality of compositions produced by the process.

Provided herein are methods for manufacturing a cell composition,comprising incubating and/or contacting an input composition comprisinga plurality of cells with one or more agents and/or under one or moreconditions thereby generating the cell composition, wherein the cellcomposition comprises one or a plurality of cells that are genetically,phenotypically, and/or functionally different from one or a plurality ofcells from the input composition, and wherein the cell compositioncomprises one or a plurality of cells that comprise a cell surfaceglycan profile comprising one or more target glycans and/or each of theone or more target glycans in the cell surface glycan profile differs byno more than 25% from the cell surface glycan profile or each of the oneor more target glycans to the total glycans present in a referencesample, wherein the cell surface glycan profile comprises glycansreleased from the surface of cells in the cell composition. In someembodiments of any of the provided methods, the cell surface glycanprofile or the one or more target glycans is determined according to anyof the methods provided herein.

In certain embodiments of any of the provided methods, the cellcomposition comprises cells comprising a recombinant nucleic acid. Inparticular embodiments of any of the provided methods, prior to, during,or subsequent to the incubation and/or contacting, the method comprisesone or more steps selected from cell washing, dilution, isolation,selection, separation, cultivation, stimulation, introduction of arecombinant nucleic acid, cryopreservation, formulation and/orpackaging. In some embodiments of any of the provided methods, prior to,during, or subsequent to incubation and/or contacting, the methodcomprises one or more steps selected from: cells isolated from abiological sample by leukapheresis or apheresis; cells selected from abiological sample by immunoaffinity-based methods; and/or cellsintroduced with a recombinant nucleic acid, optionally a viral vectorencoding a recombinant protein; and/or cell incubated in the presence ofone or more agent, optionally one more peptide, protein, polypeptide,nucleic acid, small molecule; and/or cells activated and/or expanded inthe presence of one or more stimulating conditions; and/orcryopreservation of cells in the presence of a cryoprotectant; and/orcells formulated for administration to a subject, optionally in thepresence of a pharmaceutically acceptable excipient.

In certain embodiments of any of the provided methods, he one or moretest agents or conditions comprises presence or concentration of serum;time in culture; presence or amount of a stimulating agent; the type orextent of a stimulating agent; presence or amount of amino acids;temperature; the source or cell types of the source composition; theratio or percentage of cell types in the source composition, optionallythe CD4+/CD8+ T cell ratio; the presence or amount of beads; celldensity; static culture; rocking culture; perfusion; the type of viralvector; the vector copy number; the presence of a transduction adjuvant;cell density of the source composition in cryopreservation; the extentof expression of the recombinant receptor; or the presence of a compoundto modulate cell phenotype.

In particular embodiments of any of the provided methods, the one ormore test agents or conditions comprises stimulating conditions, apeptide, a protein, a polypeptide, a nucleic acid, a small molecule,and/or a recombinant nucleic acid, optionally a viral vector encoding arecombinant protein. In some embodiments of any of the provided methods,the agent modulates the growth, proliferation, viability,differentiation, intracellular signaling, activation and/or expansion ofone or more cells in the cell composition.

In certain embodiments of any of the provided methods, the stimulatingcondition comprises incubation with a stimulatory agent capable ofactivating one or more intracellular signaling domains of one or morecomponents of a TCR complex and/or one or more intracellular signalingdomains of one or more costimulatory molecules. In particularembodiments of any of the provided methods, the stimulatory agentcomprises a primary agent that specifically binds to a member of a TCRcomplex and a secondary agent that specifically binds to a T cellcostimulatory molecule. In some embodiments of any of the providedmethods, the primary agent specifically binds to CD3 and/or thecostimulatory molecule is selected from the group consisting of CD28,CD137 (4-1-BB), OX40, or ICOS.

In certain embodiments of any of the provided methods, stimulatory agentcomprises an anti-CD3 antibody or antigen binding fragment thereofand/or an anti-CD28 antibody or an antigen-binding fragment thereto. Inparticular embodiments of any of the provided methods, the primary andsecondary agents comprise antibodies and/or are present on the surfaceof a solid support. In some embodiments of any of the provided methods,the solid support is or comprises a bead. In certain embodiments of anyof the provided methods, the stimulating conditions comprises thepresence of one or more cytokines, optionally IL-2, IL-15 and/or IL-7.

In particular embodiments of any of the provided methods, the referencesample is a reference standard comprising a release specification, alabel requirement or a compendia specification. In some embodiments ofany of the provided methods, the reference sample comprises an averageor median of the presence, absence, identity and/or level of the one ormore target glycan or glycans among a plurality of compositions producedby the incubating and/or the contacting the source composition with theone or more agents and/or under the one or more conditions. In certainembodiments of any of the provided methods, the cell surface glycanprofile comprising the one or more target glycans or each of the one ormore target glycans differs by no more than or about 20%, no more thanor about 15%, no more than or about 10% or no more than or about 5% fromthe cell surface glycan profile or each of the one or more targetglycans to the total glycans present in the reference sample. Inparticular embodiments of any of the provided methods, the targetglycans comprise at least 25, at least 50, at least 60, at least 70, atleast 80, at least 90, at least 100, or at least 200 different speciesof glycans, optionally wherein the species of glycans are N-glycans.

In some embodiments of any of the provided methods, the target glycan orglycans comprise high mannose N-glycans, bisected and Sialyl Lewis^(X)N-glycans, and/or N-acetyl lactosamine containing N-glycans. In certainembodiments of any of the provided methods, the target glycan or glycanscomprise a fucosylated biantennary complex glycan having no reducing endterminal galactose residues, a fucosylated biantennary complex glycanhaving one reducing end terminal galactose residue, a fucosylatedbiantennary complex glycan having two reducing end terminal galactoseresidues, a biantennary complex glycan having no reducing end terminalgalactose residues, a biantennary complex glycan having one reducing endterminal galactose residue, a biantennary complex glycan having tworeducing end terminal galactose residues, a fucosylated biantennarycomplex glycan having two galactose residues and one N-acetylneuraminicacid residue, a fucosylated biantennary complex glycan having twogalactose residues and two N-acetylneuraminic acid residues, abiantennary complex glycan having two galactose residues and twoN-acetylneuraminic acid residues, a high mannose glycan having fivemannose residues, a high mannose glycan having six mannose residues, ahigh mannose glycan having seven mannose residues, a high mannose glycanhaving eight mannose residues, and/or a high mannose glycan having ninemannose residues. In particular embodiments of any of the providedmethods, the target glycan or glycans comprise the glycans present inTable E1 or a subset thereof. In some embodiments of any of the providedmethods, the target glycan or glycans comprise the glycans detectable inthe cell surface glycan profile.

Provided herein are methods for screening one or more test agents orconditions on a cell composition, comprising: (a) assessing a cellsurface glycan profile in a sample from a test cell composition, whereinthe test cell composition is or is derived from an source compositionthat has been incubated or treated in the presence of one or more testagents or conditions; and (b) comparing the cell surface glycan profileof the sample to the cell surface glycan profile of a reference sample,the reference sample comprising one or more target glycans.

Also provided herein are methods for screening one or more test agentsor conditions on a cell composition, comprising comparing the cellsurface glycan profile of a sample compared to the cell surface glycanprofile of a reference sample, wherein the sample is from a test cellcomposition that is or is derived from an source composition that hasbeen incubated or treated in the presence of one or more test agents orconditions. In certain embodiments of any of the provided methods, thecell surface glycan profile comprises the presence, absence, identityand/or level of one or more glycans in the sample. In particularembodiments of any of the provided methods, the cell surface glycanprofile is determined according to the method of any of the methodsprovided herein.

In some embodiments of any of the provided methods, the reference sampleis derived from a composition incubated or treated under the same orsubstantially the same conditions as the test cell composition or sourcecomposition except in the absence of treating in the presence of the oneor more test agents or conditions or in the presence of one or morealternative test agents or conditions. In certain embodiments of any ofthe provided methods, the reference sample comprises an average ormedian of the presence, absence, identity and/or level of the one ormore target glycan or glycans among a plurality of compositionsincubated or treated in the presence of the one or more test agents orconditions. In particular embodiments of any of the provided methods,the one or more test agents or conditions comprises presence orconcentration of serum; time in culture; presence or amount of astimulating agent; the type or extent of a stimulating agent; presenceor amount of amino acids; temperature; the source or cell types of thesource composition; the ratio or percentage of cell types in the sourcecomposition, optionally the CD4+/CD8+ T cell ratio; the presence oramount of beads; cell density; static culture; rocking culture;perfusion; the type of viral vector; the vector copy number; thepresence of a transduction adjuvant; cell density of the sourcecomposition in cryopreservation; the extent of expression of therecombinant receptor; or the presence of a compound to modulate cellphenotype.

In some embodiments of any of the provided methods, the one or more testagents or conditions comprises one or more compounds from a library oftest compounds. In certain embodiments of any of the provided methods,the target glycans comprise at least 25, at least 50, at least 60, atleast 70, at least 80, at least 90, at least 100, or at least 200different species of glycans, optionally wherein the species of glycansare N-glycans. In particular embodiments of any of the provided methods,the target glycan or glycans comprise high mannose N-glycans, bisectedand Sialyl Lewis' N-glycans, and/or N-acetyl lactosamine containingN-glycans.

In some embodiments of any of the provided methods, the target glycan orglycans comprise a fucosylated biantennary complex glycan having noreducing end terminal galactose residues, a fucosylated biantennarycomplex glycan having one reducing end terminal galactose residue, afucosylated biantennary complex glycan having two reducing end terminalgalactose residues, a biantennary complex glycan having no reducing endterminal galactose residues, a biantennary complex glycan having onereducing end terminal galactose residue, a biantennary complex glycanhaving two reducing end terminal galactose residues, a fucosylatedbiantennary complex glycan having two galactose residues and oneN-acetylneuraminic acid residue, a fucosylated biantennary complexglycan having two galactose residues and two N-acetylneuraminic acidresidues, a biantennary complex glycan having two galactose residues andtwo N-acetylneuraminic acid residues, a high mannose glycan having fivemannose residues, a high mannose glycan having six mannose residues, ahigh mannose glycan having seven mannose residues, a high mannose glycanhaving eight mannose residues, and/or a high mannose glycan having ninemannose residues. In certain embodiments of any of the provided methods,the target glycan or glycans comprise the glycans present in Table E1 ora subset thereof. In particular embodiments of any of the providedmethods, the target glycan or glycans comprise the glycans detectable inthe cell surface glycan profile.

In some embodiments of any of the provided methods, the method comprisesselecting the one or more test agent or conditions for incubating ortreating the cells if the comparison indicates the cell surface glycanprofile of the sample or each of the one or more target glycans issubstantially the same as the reference sample and/or if the comparisonindicates the cell surface glycan profile comprising the one or moretarget glycans or each of the one or more target glycans differs by nomore than or about 20%, no more than or about 15%, no more than or about10% or no more than or about 5% from the cell surface glycan profile oreach of the one or more target glycans to the total glycans present inthe reference sample. In certain embodiments of any of the providedmethods, the methods comprise repeating the method with one or morefurther test agent or condition if the comparison indicates the cellsurface glycan profile of the sample or each of the one or more targetglycans is substantially different from the reference sample and/or ifthe comparison indicate the cell surface glycan profile comprising theone or more target glycans or each of the one or more target glycansdiffers by greater than or greater than about 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90% or more from the cell surface glycan profile or eachof the one or more target glycans to the total glycans present in thereference sample.

In particular embodiments of any of the provided methods, the test agentis a candidate for modulating the growth, proliferation, viability,differentiation, activation and/or expansion, of one or more cells inthe test cell composition. In certain embodiments of any of the providedmethods, the test cell composition comprises cells comprising arecombinant nucleic acid. In some embodiments of any of the providedmethods, the recombinant nucleic acid encodes a recombinant protein,optionally a recombinant receptor. In particular embodiments of any ofthe provided methods, the recombinant receptor is or comprises achimeric receptor and/or a recombinant antigen receptor.

In certain embodiments of any of the provided methods, the recombinantreceptor is capable of binding to a target antigen that is associatedwith, specific to, and/or expressed on a cell or tissue of a disease,disorder or condition. In some embodiments of any of the providedmethods, the disease, disorder or condition is an infectious disease ordisorder, an autoimmune disease, an inflammatory disease, or a tumor ora cancer. In particular embodiments of any of the provided methods, thetarget antigen is a tumor antigen. In certain embodiments of any of theprovided methods, the target antigen is selected from among ROR1, B cellmaturation antigen (BCMA), carbonic anhydrase 9 (CAIX), tEGFR, Her2/neu(receptor tyrosine kinase erbB2), L1-CAM, CD19, CD20, CD22, mesothelin,CEA, and hepatitis B surface antigen, anti-folate receptor, CD23, CD24,CD30, CD33, CD38, CD44, EGFR, epithelial glycoprotein 2 (EPG-2),epithelial glycoprotein 40 (EPG-40), EPHa2, erb-B2, erb-B3, erb-B4, erbBdimers, EGFR vIII, folate binding protein (FBP), FCRLS, FCRHS, fetalacetylcholine receptor, GD2, GD3, HMW-MAA, IL-22R-alpha, IL-13R-alpha2,kinase insert domain receptor (kdr), kappa light chain, Lewis Y, L1-celladhesion molecule, (L1-CAM), Melanoma-associated antigen (MAGE)-A1,MAGE-A3, MAGE-A6, Preferentially expressed antigen of melanoma (PRAME),survivin, TAG72, B7-H6, IL-13 receptor alpha 2 (IL-13Rα2), CA9, GD3,HMW-MAA, CD171, G250/CAIX, HLA-AI MAGE A1, HLA-A2 NY-ESO-1, PSCA, folatereceptor-a, CD44v6, CD44v7/8, avb6 integrin, 8H9, NCAM, VEGF receptors,5T4, Foetal AchR, NKG2D ligands, CD44v6, dual antigen, a cancer-testesantigen, mesothelin, murine CMV, mucin 1 (MUC1), MUC16, PSCA, NKG2D,NY-ESO-1, MART-1, gp100, oncofetal antigen, ROR1, TAG72, VEGF-R2,carcinoembryonic antigen (CEA), Her2/neu, estrogen receptor,progesterone receptor, ephrinB2, CD123, c-Met, GD-2, O-acetylated GD2(OGD2), CE7, Wilms Tumor 1 (WT-1), a cyclin, cyclin A2, CCL-1, CD138, apathogen-specific antigen and an antigen associated with a universaltag. In some embodiments of any of the provided methods, the recombinantreceptor is or comprises a functional non-TCR antigen receptor or a TCRor antigen-binding fragment thereof. In certain embodiments of any ofthe provided methods, the recombinant receptor is a chimeric antigenreceptor (CAR).

In particular embodiments, the target antigen is selected from amongαvβ6 integrin (avb6 integrin), B cell maturation antigen (BCMA), B7-H3,B7-H6, carbonic anhydrase 9 (CA9, also known as CAIX or G250), acancer-testis antigen, cancer/testis antigen 1B (CTAG, also known asNY-ESO-1 and LAGE-2), carcinoembryonic antigen (CEA), a cyclin, cyclinA2, C-C Motif Chemokine Ligand 1 (CCL-1), CD19, CD20, CD22, CD23, CD24,CD30, CD33, CD38, CD44, CD44v6, CD44v7/8, CD123, CD133, CD138, CD171,chondroitin sulfate proteoglycan 4 (CSPG4), epidermal growth factorprotein (EGFR), truncated epidermal growth factor protein (tEGFR), typeIII epidermal growth factor receptor mutation (EGFR vIII), epithelialglycoprotein 2 (EPG-2), epithelial glycoprotein 40 (EPG-40), ephrinB2,ephrine receptor A2 (EPHa2), estrogen receptor, Fc receptor like 5(FCRLS; also known as Fc receptor homolog 5 or FCRHS), fetalacetylcholine receptor (fetal AchR), a folate binding protein (FBP),folate receptor alpha, ganglioside GD2, O-acetylated GD2 (OGD2),ganglioside GD3, glycoprotein 100 (gp100), glypican-3 (GPC3), G ProteinCoupled Receptor 5D (GPCRSD), Her2/neu (receptor tyrosine kinaseerb-B2), Her3 (erb-B3), Her4 (erb-B4), erbB dimers, Human high molecularweight-melanoma-associated antigen (HMW-MAA), hepatitis B surfaceantigen, Human leukocyte antigen A1 (HLA-A1), Human leukocyte antigen A2(HLA-A2), IL-22 receptor alpha(IL-22Rα), IL-13 receptor alpha 2(IL-13Rα2), kinase insert domain receptor (kdr), kappa light chain, L1cell adhesion molecule (L1-CAM), CE7 epitope of L1-CAM, Leucine RichRepeat Containing 8 Family Member A (LRRC8A), Lewis Y,Melanoma-associated antigen (MAGE)-A1, MAGE-A3, MAGE-A6, MAGE-A10,mesothelin (MSLN), c-Met, murine cytomegalovirus (CMV), mucin 1 (MUC1),MUC16, natural killer group 2 member D (NKG2D) ligands, melan A(MART-1), neural cell adhesion molecule (NCAM), oncofetal antigen,Preferentially expressed antigen of melanoma (PRAME), progesteronereceptor, a prostate specific antigen, prostate stem cell antigen(PSCA), prostate specific membrane antigen (PSMA), Receptor TyrosineKinase Like Orphan Receptor 1 (ROR1), survivin, Trophoblast glycoprotein(TPBG also known as 5T4), tumor-associated glycoprotein 72 (TAG72),Tyrosinase related protein 1 (TRP1, also known as TYRP1 or gp75),Tyrosinase related protein 2 (TRP2, also known as dopachrometautomerase, dopachrome delta-isomerase or DCT), vascular endothelialgrowth factor receptor (VEGFR), vascular endothelial growth factorreceptor 2 (VEGFR2), Wilms Tumor 1 (WT-1), a pathogen-specific orpathogen-expressed antigen, or an antigen associated with a universaltag, and/or biotinylated molecules, and/or molecules expressed by HIV,HCV, HBV or other pathogens.

In particular embodiments of any of the provided methods, the cellscomprise mammalian cells or the test cell composition comprisesmammalian cells. In some embodiments of any of the provided methods, thecells comprise human cells or the test cell composition comprises humancells. In certain embodiments of any of the provided methods, the cellscomprise stem cells or the test cell composition comprises stem cells.In particular embodiments of any of the provided methods, the stem cellis an induced pluripotent stem cell (iPSC). In some embodiments of anyof the provided methods, the composition or test cell compositioncomprises cells present in an apheresis product or a leukapheresisproduct or cells derived therefrom.

In certain embodiments of any of the provided methods: the cellscomprise immune cells, white blood cells, peripheral blood mononuclearcells (PBMC), lymphocytes, or unfractionated T cells; or the test cellcomposition comprises immune cells, white blood cells, peripheral bloodmononuclear cells (PBMC), lymphocytes, or unfractionated T cells. Inparticular embodiments of any of the provided methods, the cellscomprise an immune cell or the test cell composition comprises immunecells. In some embodiments of any of the provided methods, the immunecell is a T cell, B cell, macrophage, neutrophil, natural killer (NK)cell or dendritic cell. In certain embodiments of any of the providedmethods, the cells comprise T cells that are CD4+ and/or CD8+ T cells orthe test cell composition comprises T cells that are CD4+ and/or CD8+ Tcells. In particular embodiments of any of the provided methods, thecells are primary cells.

Particular embodiments provide a method of detecting a presence,absence, identity, and/or level of one or more substances in a cellcomposition, the method comprising: (a) assessing the cell surfaceglycan profile in a sample from a test cell composition comprising aplurality of cells according to any of the methods provided herein,wherein the plurality of cells are from or are derived from a cell type;and (b) identifying one or more non-native glycans in the cell surfaceglycan profile that are not synthesized and/or expressed by cells of thecell type. In certain embodiments, the cell type is human. In someembodiments, the cell type is an immune cell. In particular embodiments,the cell type is a T cell.

In certain embodiments, the test cell composition was produced byculturing and/or incubating the plurality of cells in the presence of asubstance, in which comprises at least one protein comprising one ormore non-native glycans. In some embodiments, the at least one proteinis an albumin, a growth factor, a cytokine, a chemokine, an insulin orinsulin-like peptide, a transferrin, or a superoxide dismutase. Inparticular embodiments, the at least one protein is a recombinantprotein. In certain embodiments, the one or more non-native glycansand/or the at least one protein are present in a serum. In someembodiments, the serum is fetal bovine serum (FBS), bovine calf serum(BCS), newborn calf serum (NBCS), horse serum, goat serum, lamb serum,donkey serum, or porcine serum.

In particular embodiments, the one or more non-native glycans and/or theat least one protein are (i) not produced by and/or (ii) not expressedon the surface of cells from the same order, family, genus, or speciesas the cells of the plurality. In certain embodiments, the one or morenon-native glycans comprise a non-human glycan. In some embodiments, theidentifying the one or more non-native glycan comprises comparing thesurface glycan profile to a reference glycan profile, wherein thereference sample is a glycan profile from a source containing the one ormore non-native glycans. In particular embodiments, the reference glycanprofile is generated from a reference sample comprising a substance,that has not been contacted, incubated, and/or exposed to the cells ofthe plurality. In some embodiments, the reference glycan profile isgenerated from a reference sample comprising a media, a serum, or acomponent thereof, or from a protein or recombinant protein thereof,that has not been contacted, incubated and/or exposed to the cells ofthe plurality.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B depict exemplary methods for generating surface N-glcyanprofiles. FIG. 1A depicts an overview of an exemplary method for mappingcell surface N-glycans. In the method, a composition containing whole,intact cells is incubated with PNGaseF to release N-glycans. The cellsare removed and the sample is subjected to solid phase extraction (SPE),followed by separation of glycans using hydrophilic interactionchromatography (HILIC) liquid chromatography (LC) and detection byfluorescence (Waters ACQUITY I-Class) and positive electrosprayionization (ESI) mass spectrometry (MS), Q-Exactive™ HF (ThermoScientific)) for relative quantification and identification. FIG. 1Bshows a comparison of methods for traditional targeted analysis byimmunoaffinity and flow cytometry (Targeted) with the exemplary methodprovided herein (unbiased).

FIG. 2 shows a surface N-glycan map of released N-glycans followingPNGase F enzyme treatment of whole intact cells from a composition ofCD8+ T cells containing cells expressing an anti-CD19 chimeric antigenreceptor (CAR). Specifically, FIG. 2 depicts an annotated chromatogramof N-glycans separated using HILIC liquid chromatography and detected byfluorescence (HILIC-FLR).

FIGS. 3A and 3B show exemplary readouts of surface N-glycan profiles.FIG. 3A shows an HILIC-FLR of PNGase F released N-glycans from acomposition containing CD4+/CD8+ T cells expressing an anti-CD19 CAR.FIG. 3B shows a total ion chromatogram (TIC) from the same cellcomposition as FIG. 3A.

FIGS. 4A-C show chromatograms produced by HILIC-LC and tandem MS ofN-glycans releases from whole intact CD3+ activated T cells followingPNGase F treatment. FIG. 4A shows a HILIC-FLR chromatogram of PNGase Freleased N-glycans from the activated CD3+ T cell composition. FIG. 4Bshows an extracted ion chromatogram (XIC) produced from the first stageof the tandem MS for the exemplary N-glycan, A3S3F (theoretical mass of1113.0933), in the +3 charged state using a 5 ppm mass tolerance. FIG.4C shows the MS/MS fragmentation of a further exemplary N-glycan, A3S4F(theoretical mass of 1210.4614), produced by the second stage of thetandem MS. Dashed boxes in FIG. 4C indicate different n-acetylglucosamine residue linkages.

FIG. 5A-5C show overlays of peaks corresponding to surface N-glycansvisualized by fluorescence detection and mass spectrometry from samplestaken from cell compositions collected at different stages of productionof a therapeutic cell composition containing cells expressing ananti-CD19 CAR. Surface N glycan maps of CD4+ and CD8+ T cellcompositions of cells obtained from immunoaffinity-based selection ofcells from leukapheresis samples, cells that were activated andtransduced with a viral vector encoding an anti-CD19 CAR, and cells thatwere transduced, expanded, and harvested as a cryopreserved drug productare shown. FIG. 5A shown an overlay of high mannose N-glycans in samplesfrom CD4+ and CD8+ T cell compositions. On both the CD4+ and CD8+overlays, where the X-axis is marked with an arrow, the top linedisplays the N-glycan profile of the cryopreserved drug product, themiddle line displays the N-glycan profile of the cells that wereactivated and transduced, and the bottom line shows the N-glycan profileof the cells obtained from immunoaffinity-based selection.

FIG. 5B shows an overlay of bisected N-glycans in samples from CD4+ andCD8+ T cell compositions. On both the CD4+ and CD8+ overlays, where theX-axis is marked with an arrow, the top line displays the N-glycanprofile of the cryopreserved drug product, the middle line) displays theN-glycan profile of the cells that were activated and transduced, andthe bottom line shows the N-glycan profile of the cells obtained fromimmunoaffinity-based selection. FIG. 5C shows an overlay ofpolylactosamine N-glycans in samples from CD4+ and CD8+ T cellcompositions. On both the CD4+ and CD8+ overlays, where the X-axis ismarked with an arrow, the top line shows the N-glycan profile of thecells obtained from immunoaffinity-based selection, the middle linedisplays the N-glycan profile of the cryopreserved drug product, and thebottom line displays the N-glycan profile of the cells that wereactivated and transduced.

FIG. 6 shows a comparison of surface N-linked glycan profiles of cellcompositions collected at different stages of production of a cellcomposition containing cells expressing an anti-CD19 CAR: mononuclearcells (top); CD3+ T cells (middle); and activated CD3+ T cells (bottom).Exemplary differences are highlighted with annotation for increased (++or +), amount (n), or decreased (−) amount of glycan species. Peakscorresponding to the A2S1FB and A2S2FB N-glycans are indicated by asolid line. Regions of the chromatogram corresponding tobiantannary/hybrid and N-acetyl lactosamine repeat N-glycans areindicated by dashed lines.

FIG. 7 shows surface N-glycan maps visualized by fluorescence detectionand mass spectrometry of surface N-glycan samples from differentCAR-expressing T cell compositions, including CD4+ and CD8+ T cellcompositions expressing an anti-CD19 CAR, a T cell compositionexpressing an alternative anti-CD19 CAR or a T cell compositionexpressing an CAR that recognizes an alternative target antigen.

FIG. 8 shows surface N-glycan profiles visualized by fluorescencedetection and mass spectrometry from CD4+ T cell compositions containinganti-CD19 CAR+ T cells produced by either an exemplary or alternativeengineering process. Surface N-glycan profiles from an anti-CD19CAR-expressing CD4+ T cell composition produced by the alternativeengineering process (top panel), an anti-CD19 CAR-expressing CD4+ T cellcomposition engineered from cells from the same individual subjectproduced by the exemplary engineering process (middle panel), and ananti-CD19 CAR-expressing T cell composition engineered from cells of adifferent individual subject produced by the alternative engineeringprocess (bottom panel). Arrows indicate peaks associated with specificN-glycans; “B” indicates a bisected N-glycan and “SL” indicates a SialylLewis^(X) N-glycan.

FIG. 9 FIG. 10 shows surface N-glycan profiles visualized byfluorescence detection and mass spectrometry from three exemplarydifferent anti-CD19 CAR-expressing CD8+ T cell compositions that weregenerated by substantially the same process, but that differed in twoconditions involved in cultivating the cells. Arrows indicate a peakassociated with a bisected N-glycan.

FIG. 10 shows surface N-glycan profiles visualized by fluorescencedetection and mass spectrometry from three exemplary different anti-CD19CAR-expressing CD4+ T cell compositions that were generated bysubstantially the same process, but that differed in two conditionsinvolved in cultivating the cells. Arrows indicate a peak associatedwith a bisected N-glycan.

FIG. 11 shows surface N-glycan profiles visualized by fluorescencedetection and mass spectrometry of surface N-glycan samples fromdifferent anti-CD19 CAR-expressing CD8+ T cell compositions. SurfaceN-glycan profiles from anti-CD19 CAR-expressing CD8+ T cell compositionsproduced from cells from healthy subjects (top and middle panels) andfrom a subject with disease associated with CD10 (bottom panel) areshown. Arrows indicate peaks associated with specific N-glycans; “B”indicates a bisected N-glycan and “SL” indicates a Sialyl Lewis^(X)N-glycan.

FIG. 12 shows surface N-glycan profiles visualized by fluorescencedetection and mass spectrometry from different T cell compositionscontaining T cells that express a CAR with an alternative targetantigen. Surface N-glycan profiles from CAR-expressing T cellcompositions produced from cells from healthy subjects (top and bottompanels) and from a subject with a disease associated with the targetantigen (middle panel) are shown. Arrows indicate peaks associated withspecific N-glycans; “B” indicates a bisected N-glycan and “SL” indicatesa Sialyl Lewis' N-glycan.

FIG. 13 shows surface N-glycan profiles visualized by fluorescencedetection and mass spectrometry from a sample of fetal bovine serum(FBS; top panel) and surface N-glycan profiles from a CD4+ anti-CD19CAR-expressing CD4+ T cell composition engineered from cells from thesame individual subject produced by the two different engineeringprocesses (middle and bottom panels). Arrows indicate peaks associatedwith FBS N-glycans; “AG” indicates a peak associated with a non-humanalpha-gal N-glycan and “NGNA” indicates a peak associated with anon-human N-glycolylneuraminic acid (NGNA)N-glycan.

DETAILED DESCRIPTION

Provided herein are methods for assessing glycans that are expressedand/or exposed on the cell surface, i.e., surface glycans, bydetermining the presence, absence, or level of glycans present in asample containing glycans that are or have been released from thesurface of cells in a composition. In some embodiments, the providedmethods include one or more steps for releasing glycans, e.g.,N-glycans, from the surface of cells in the composition. In certainembodiments, the methods provided herein provide steps for detectingglycans that have been released from the cell surface by one or moresuitable techniques, for example one or more of liquid chromatographyand mass spectrometry. In some embodiments, the methods provided hereinallow for a highly sensitive and accurate assessment or analysis of thesurface glycan expression profile of the cell composition.

In particular embodiments, surface glycans of a population orcomposition of cells can be assessed and/or analyzed by the methodsprovided herein. In certain embodiments, the methods provide steps forincubating the cells under conditions suitable for releasing glycansfrom the surface of cells. In certain embodiments, the conditionsinclude contacting, treating, and/or incubating the cells with an agentthat promotes and/or catalyzes the release of glycans from the surfaceof the cells, for example by removing intact glycans from proteins orother moieties that are present, expressed, and/or exposed at the cellsurface. In certain embodiments, the methods provided herein alsoinclude steps for derivatizing and/or labeling glycans to enhance orincrease sensitivity and/or accuracy for identification, measurement,and/or detection of the glycans. In particular embodiments, the providedmethods include steps for detecting the glycans, e.g., labeled glycans,through combined techniques of liquid chromatography and massspectrometry, allowing for highly accurate and sensitive measurements ofthe presence, identity, and levels of individual glycans present in thesample of released surface glycans.

Also provided herein are methods of assaying a cell composition byassessing the cell surface glycan profile of the cell composition andcomparing it to a reference sample. For example, in some embodiments, acell composition, e.g., a therapeutic cell composition, may have adistinct profile of glycan expression with respect to the level ofsurface expression of distinct glycan species, types of glycans, and/orglycan families as compared to compositions of different cells. Thus, insome embodiments, a reference standard may be generated by assessing aplurality of cell compositions. Such a reference standard may be used,in some embodiments, as a quality control and/or for a release assay, orto monitor or control the manufacture or culture of the cellcompositions.

In addition, provided herein are methods for generating a plurality ofcell compositions, e.g., therapeutic cell compositions and/orcompositions containing engineered cells, with low variability withrespect to surface glycan expression. In some embodiments, the leveland/or expression of distinct glycan species, types of glycans, and/orglycan families at the cell surface may contribute or at least correlateto different aspects of cell physiology or function. Thus, in someembodiments, it is desirable to produce cell compositions with a highdegree of similarity with respect to surface glycan expression.

Particular embodiments contemplate that cell therapies, and inparticular adoptive T-Cell therapies, represent a powerful technologyfor the treatment, alleviation, and/or amelioration of various diseases,such as cancer. Current analytical tools available for assessingtherapeutic or pharmaceutical cell compositions include examination ofcell surface or internal markers via flow cytometry. In someembodiments, the methods provided herein complement and/or enhancecurrent methods for evaluating cell compositions, which representmethods and techniques to reliably evaluate characteristics oftherapeutic or pharmaceutical cell compositions as well as cells atdifferent stages of a manufacturing or development process. Inparticular embodiments, provided herein are unbiased methods useful forreleasing cell surface N-linked glycans and for detecting, identifying,and/or quantifying one or more individual N-glycan species (such, forexample, methods exemplified by FIGS. 1A-1B).

In certain embodiments, the methods provided herein provide informationregarding the presence, absence, identities, relative amounts, andlevels of individual glycan species that are expressed on a cellsurface. Thus, in some embodiments, the methods provided herein aresuitable for generating a glycan expression profile of a cellcomposition. In particular embodiments, the methods provided hereingenerate such profiles with a higher resolution and degree of accuracythan existing methods for monitoring surface glycan expression in cells.For example, in some embodiments, the methods provided herein detectindividual glycan species that are present in lower amounts within asample than existing methods, allowing for the detection and/ormonitoring of more individual glycans present in a cell composition thanwould be allowed by available techniques.

In some embodiments, the methods provided herein provide one or moresteps whereby glycans are directly released from the cell surface, e.g.,from proteins expressed and/or localized at the cell surface. This is incontrast to many existing methods, which rely on a prior step of lysingthe cells and/or purifying proteins, e.g., membrane proteins, prior to astep of releasing glycans from their respective glycoconjugate. In someembodiments, one advantage of the presently claimed methods is that bystimulating the release of surface glycans from intact cells, onlysurface exposed glycans are collected. Glycans, including O-glycans andN-glycans, are also present internally in cells, for example expressedon nascent polypeptides processed in the Golgi apparatus, or asadditions or modifications on signaling molecules. Therefore, glycanscollected by other methods that involve steps of lysing cells and/orobtaining cell homogenates may contain mixtures of internally andexternally localized glycans, or at the very least, include somefraction of glycans that were not expressed on the surface. In contrast,particular embodiments of the methods provided herein collect a distinctpool of glycans that were expressed on the surface.

In some embodiments, an additional advantage of releasing glycansdirectly from the cell surface, as opposed, for example, to methods thatinvolve lysing cells, is that the cells remain intact even after theglycans have been removed. Thus, in some embodiments, cells from thesame composition may be further analyzed with additional techniquesafter the glycans have been removed. In some embodiments, cells areprocessed for further analysis after surface glycans are removed, e.g.,microarray or proteomics.

In particular embodiments, the methods provided herein remove surfaceglycans without exposing the cell surface to proteases, such as trypsin.In particular embodiments, one advantage of avoiding the use ofproteases to remove surface glycans is that the surface exposed proteinsremain intact. In some embodiments, this may allow for further analysisof the cells, such as by flow cytometry, where the removal of surfaceglycans may expose additional targets for sorting, or allow for surfaceexposed proteins to be isolated and further analyzed. For example,enzymatic release of N-glycans attached to the nitrogen of asparagine,thereby converting asparagine to aspartate. In certain embodiments,another advantage of avoiding proteases to remove surface glycans isthat particular glycans may be selectively removed by selection of theappropriate agents or conditions. For example, an agent may be chosenthat selectively releases N-glycans while leaving O-glycans attached atthe cell surface, a selectivity that might not be achieved by digestingsurface proteins.

In certain embodiments, the methods provided herein provide a means todetect surface glycan expression with a high degree of accuracy andsensitivity that is not achieved by existing methods. Thus, in someembodiments, the methods provided herein provide a means to assess thephysiology and functionality of cell compositions, including therapeuticand pharmaceutical cell compositions.

In some aspects, the provided methods permit characterization of thecell surface glycome of any cell preparation or composition. Becauseglycosylation has been implicated in cell signaling, adhesion, homingproperties and other functional activities or properties, it iscontemplated that differences in the cell surface glycome may provide auseful tool for analyzing and assessing functional differences betweenand among cell compositions. In some aspects, the methods can be used toassess features of cell therapies, such as chimeric antigen receptor(CAR+) T cell therapies, including cell therapies involving multiple exvivo processing steps before infusion into a subject. In some aspects,the various processes involved in engineering a cell therapy, includingbut not limited to, cell isolation, selection, cryopreservation,stimulation, activation, transduction, expansion and/or formulation, mayimpact or alter one or more features of a therapeutic cell composition.

In some embodiments, however, the current analytical tools for studyingand evaluating cell compositions, including therapeutic cellcompositions, are primarily confined to targeted examination of cellsurface or internal markers via flow cytometry. As an alternative, theprovided methods permit the generation of a robust glycan map, includinghigh resolution chromatography and, in some cases, combined with highresolution mass spectrometry to allow for detailed characterization andidentification of surface N-glycans. In some embodiments, the methodsherein provide an unbiased technique to characterize, investigate,and/or evaluate cell compositions, including therapeutic cellcompositions, including impacts of different processes or featuresbetween and among different cell compositions.

All publications, including patent documents, scientific articles anddatabases, referred to in this application are incorporated by referencein their entirety for all purposes to the same extent as if eachindividual publication were individually incorporated by reference. If adefinition set forth herein is contrary to or otherwise inconsistentwith a definition set forth in the patents, applications, publishedapplications and other publications that are herein incorporated byreference, the definition set forth herein prevails over the definitionthat is incorporated herein by reference.

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described.

I. ASSESSING CELL SURFACE GLYCAN EXPRESSION PROFILES

Provided herein are methods of identifying, quantifying, and/oranalyzing glycans, e.g., N-glycans, that are expressed on the surface ofcells. In certain embodiments, the presence, absence, and relativeabundance of individual glycan species are detected with high resolutionand sensitivity. In certain embodiments, the methods include proceduresand/or modifications to improve the detection of the glycans. In certainembodiments, the detection of the glycans may be performed by atechnique capable of identifying and/or quantifying amounts ofindividual species of glycans. In certain embodiments, a species ofglycans includes glycans that have identical structures that aredifferent from the structures of other glycan species. In particularembodiments, the technique is a mass spectrometry technique and/or aliquid chromatography (LC) technique, such as high performance liquidchromatography (HPLC) or ultra performance liquid chromatography (UPLC).

A. Treating the Cells

In certain embodiments, a composition of cells is incubated, cultured,or treated under conditions suitable to remove, release, or detachglycans, e.g., N-glycans, from the surface of the cells of thecomposition. In certain embodiments, a composition of cells is treated,incubated, and/or contacted with an agent to remove, separate, or detachglycans, e.g., N-glycans, from the surface of the cells. In particularembodiments, the cells are intact, i.e., the cells are not lysed orhomogenized prior to treatment with the agent. In certain embodiments,the cells are live cells. In some embodiments, treating, incubating,and/or contacting the cells with the agent does not disrupt and/orrupture the cell membrane. In some embodiments, the cells are livecells, and treating, incubating, or contacting the cells with the agentdoes not kill the cells. In certain embodiments, the cells are livecells, and treating, incubating, or contacting the cells with the agentdoes not induce cell death, e.g., apoptosis or necrosis in the cells.

In some embodiments the cells are washed and/or rinsed prior to theglycan digestion. In some embodiments, the cells are washed and/orrinsed by removing media, e.g., cell culture media, from the cellsand/or adding a solution such as a fresh solution, e.g., a solution thathas not previously been contacted or exposed to the cells. In someembodiments, the solution is a buffer and/or media. Suitable buffers forwashing and/or rinsing cells are known, and include those that do notlyse cells and/or do not otherwise kill live cells. In some aspects,suitable solutions include, but are not limited to, saline solution,citrate buffer, Dulbecco's Phosphate Buffered Saline (DPBS), Earle'sBalanced Salt Solution (EBSS), Gey's balanced salt solution, Hanks'Balanced Salt Solution (HBSS), HEPES(N-2-hydroxyethylpiperazine-N′-2-ethanesulphonic acid) buffer,Krebs-Heneseleit buffer solution, Krebs-Ringer solution, MES buffer,MOPS buffer, phosphate buffer, phosphate buffered saline (PBS), Ringersolution, Tris buffer, Trizma buffer, Tyrode's solution, ormodifications or variations thereof. In particular aspects, suitablemedia is or includes, but is not limited to BME, DMEMF12, DMEMF12, DMEM,DMEM High Glucose, F-12, F-12K, ES Qualified DMEM, GMEM, IDIM, Iscove'sModified DMEM, McCoy's 5A, MEM, RPMI, StemXvivo, Xvivo, or variations ormodifications thereof.

In some embodiments, the rinsing or washing includes centrifugation. Insome aspects, the cells are transferred into a container, e.g., a vialor tube, and are centrifuged at a low speed, e.g., a speed that does notdamage or kill the cells. In some embodiments, the centrifugationpellets the cells. In certain embodiments, after the centrifugation, thesupernatant is removed from the cell pellet. In particular embodiments,the centrifugation is performed for, for about, or for less than 60minutes, 30 minutes, 15 minutes, 10 minutes, 5 minutes 4 minutes, 3minutes, 2 minutes, 90 seconds, 60 seconds, 30 seconds, or 15 seconds.In certain embodiments, the centrifugation is performed at, at about, orat less than 16,000×g, 12,000×g, 10,000×g, 8,000×g, 6,000×g, 5,000×g,4,000×g, 3,000×g, 2,000×g, 1,000×g, 800×g, 600×g, 500×g, 400×g, 300×g,200×g, 100×g, or 50×g. In certain embodiments, the cells are centrifugedfor or for about 3 minutes at or at about 500×g. In particularembodiments, the cells are centrifuged for or for about 3 minutes at orat about 300×g. After centrifugation, the supernatant may be removed anda fresh solution may be added and/or contacted to the cells. Optionallythe pellet may be disrupted or agitated, such as by vortex or pipetmixing, to disperse the cells into the solution.

In some embodiments, the cells are rinsed in solution, e.g., freshsolution, such as with a suitable buffer or media once, twice, threetimes, four times, five times, six times, seven times, eight times, ninetimes, ten times, or more than ten times prior to the glycan digestion.

In some embodiments, the cells are treated and/or incubated with anenzyme prior to glycan digestion. In some embodiments, the cells aretreated with an enzyme to remove or digest extracellularpolynucleotides, e.g., RNA and DNA molecules. In certain embodiments,the enzyme is a nuclease. In particular embodiments, the enzyme isbenzonase.

In some embodiments, a composition of cells that is incubated, cultured,or treated under conditions suitable to remove, release, or detachglycans, e.g., N-glycans, from the surface of the cells is a test cellcomposition or test composition. In particular embodiments, the testcell composition or test composition is a population and/or a pluralityof cells from a source composition. In some cases, the test compositionis a sample of the source composition, e.g. has been removed from asource composition containing a larger composition of the cells. In suchembodiments, the cells in the test cell composition are identical orsubstantially identical to the source composition except that the testcomposition generally contains a smaller volume and/or number orabsolute number of the cells. In some embodiments, the test cellcomposition is of a sufficient size to carry out the provided methods,and does not substantially interfere with or affect features of thesource composition. In particular embodiments, the source composition isa therapeutic cell composition or is a cell composition that is at astage or step within a process to manufacture a therapeutic cellcomposition.

In certain embodiments, the composition of cells, e.g. test cellcomposition, that is treated with the agent, such as N-glycosidase,e.g., PNGase F, contains between 1×10³ cells and 1×10¹² cells, between1×10⁴ cells and 1×10⁸ cells, between 1×10⁵ cells and 1×10⁷ cells,between 1×10⁶ cells and 1×10⁷ cells, or between 1×10⁶ cells and 5×10⁶cells. In some embodiments, the composition of cells, e.g. test cellcomposition, contains at least or at least about 1×10³ cells, at leastor at least about 5×10³ cells, at least or at least about 1×10⁴ cells,at least or at least about 5×10⁴ cells, at least or at least about 1×10⁵cells, at least or at least about 5×10⁵ cells, at least or at leastabout 6×10⁵ cells, at least or at least about 7×10⁵ cells, at least orat least about 8×10⁵ cells, at least or at least about 9×10⁵ cells, atleast or at least about 1×10⁶ cells, at least or at least about 2×10⁶cells, at least or at least about 3×10⁶ cells, at least or at leastabout 4×10⁶ cells, at least or at least about 5×10⁶ cells, at least orat least about 6×10⁶ cells, at least or at least about 7×10⁶ cells, atleast or at least about 8×10⁶ cells, about 9×10⁶ cells, at least or atleast about 1×10⁷ cells, at least or at least about 5×10⁷ cells, atleast or at least about 1×10⁸ cells, at least or at least about 5×10⁸cells, at least or at least about 1×10⁹ cells, at least or at leastabout 1×10¹⁰ cells, at least or at least about 1×10¹¹ cells, or at leastor at least about 1×10¹² cells. In certain embodiments, the compositionof cells, e.g. test cell composition, contains between 1×10⁶ cells and5×10⁶ cells. In particular embodiments, the composition of cells, e.g.test cell composition, contains between 1×10⁶ cells and 2.5×10⁶.

In some embodiments, the composition of cells, e.g. test cellcomposition, incubated with the agent, such as N-glycosidase (e.g.PNGase F) has a concentration of between 1×10³ cells/mL and 1×10¹²cells/mL, between 1×10⁴ cells/mL and 1×10⁸ cells/mL, between 1×10⁵cells/mL and 1×10⁷ cells/mL, or between 1×10⁶ cells/mL and 1×10⁷cells/mL. In particular embodiments, the composition of cells, e.g. testcell composition, has a concentration of at least or at least about5×10⁴ cells/mL, at least or at least about 1×10⁵ cells/mL, at least orat least about 5×10⁵ cells/mL, at least or at least about 6×10⁵cells/mL, at least or at least about 1×10⁶ cells/mL, at least or atleast about 5×10⁶ cells/mL, at least or at least about 5×10⁶ cells/mL,at least or at least about 6×10⁶ cells/mL, at least or at least about7×10⁶ cells/mL, at least or at least about 8×10⁶ cells/mL, at least orat least about 9×10⁶ cells/mL, at least or at least about 1×10⁷cells/mL, at least or at least about 1.25×10⁷ cells/mL, at least or atleast about 2.5×10⁷ cells/mL, at least or at least about 5×10⁷ cells/mL,at least or at least about 1×10⁸ cells/mL, at least or at least about5×10⁸ cells/mL, at least or at least about 1×10⁹ cells/mL, at least orat least about 1×10¹⁰ cells/mL, at least or at least about 1×10¹¹cells/mL, or at least or at least about 1×10¹² cells/mL. In someembodiments, the composition, e.g. test cell composition, has aconcentration of cells of between 5×10⁶ cells/mL and 2.5×10⁷ cells/mL.In certain embodiments, the composition of cells, e.g. test cellcomposition, has a concentration of between 5×10⁶ cells/mL and 1.25×10⁷cells/mL. In certain embodiments, the composition, e.g. test cellcomposition, contains a concentration of at least or at least about orabout 5×10⁶ cells/mL. In particular embodiments, the composition, e.g.test cell composition, contains a concentration of at least or at leastabout or about 1.25×10⁷ cells/mL. In some embodiments, the composition,e.g. test cell composition, contains a concentration of at least or atlast about or about 2.5×10⁷ cells/mL.

In some embodiments, the incubation, culture or treatment is carried outin a total volume of from or from about 0.005 mL to 50 mL, 0.005 mL to25 mL, 0.005 mL to 10 mL, 0.005 mL to 5 mL, 0.005 mL to 1 mL, 0.005 mLto 0.5 mL, 0.005 mL to 0.05 mL, 0.05 mL to 50 mL, 0.05 mL to 35 mL, 0.05mL to 10 mL, 0.05 mL to 5 mL, 0.05 mL to 1 mL, 0.05 mL to 0.5 mL, 0.5 mLto 50 mL, 0.5 mL to 25 mL, 0.5 mL to 10 mL, 0.5 mL to 5 mL, 0.5 mL to 1mL, 1 mL to 50 mL, 1 mL to 25 mL, 1 mL to 10 mL, 1 mL to 5 mL, 5 mL to50 mL, 5 mL to 25 mL, 5 mL to 10 mL, 10 mL to 50 mL, 10 mL to 25 mL or25 mL to 50 mL. In some embodiments, the incubation, culture ortreatment is carried out in a total volume of at least or at least about0.005 mL, 0.01 mL, 0.05 mL, 0.1 mL, 0.2 mL, 0.3 mL, 0.4 mL, 0.5 mL, 1mL, 2.0 mL, 5 mL, 10 mL, 20 mL, 30 mL, 40 mL or 50 mL.

1. Agent, e.g. N-Glycosidase

In some embodiments, the composition of cells is treated, incubated,and/or contacted with an agent, e.g. N-glycosidase, e.g. PNGase F,resulting in a removal, separation, and/or detachment of glycans, e.g.,N-glycans, from surface exposed glycoconjugate. In some embodiments, theglycoconjugate is a protein, e.g., a glycoprotein. In particularembodiments, the treating, contacting, and/or incubating the compositionof the cells with the agent results in the removal, separation, and/ordetachment of glycans from a surface exposed protein. In particularembodiments, the released, removed, and/or detached N-glycans areintact. In some embodiments, the removal, separation, and/or detachmentof the glycans from the surface exposed protein does not damage, digest,and/or otherwise alter the structure of the glycan. In particularembodiments, the removal, separation, and/or detachment of the glycansfrom the surface exposed protein does not damage, digest, and/orotherwise alter the structure of the moiety, e.g., protein, from whichthe glycan has been released. In some embodiments, the removal,separation, and/or detachment of the glycans from the surface exposedprotein results in the conversion of asparagine to aspartate, but doesnot otherwise damage, digest, and/or alter the structure of the proteinfrom which the glycan has been released.

In certain embodiments, the agent removes a glycan from aglycoconjugate, e.g., a surface exposed glycoprotein. In someembodiments, the glycan is a polysaccharide. In certain embodiments, theglycan is a branched glycan, a linear glycan, an N-linked glycan, anO-linked glycan, or a combination thereof. In certain embodiments, theagent removes an N-glycan from a glycoconjugate. In particularembodiments, N-glycans are covalently attached to proteins at asparagine(Asn) residues by an N-glycosidic bond, most commonly anN-acetylglucosamine to asparagine (GlcNAcβ1-Asn). In certainembodiments, N-glycans are attached to asparagines via anN-acetylglucosamine (“GlcNAc”) residue in an Asn-Xxx-(Ser, Thr) motif,where Xxx can be any amino acid except proline.

In certain embodiments, all N-glycans share a common core sugarsequence, Manα1-6(Manα1-3)Manβ1-4GlcNAcβ1-4GlcNAcβ1-Asn-X-Ser/Thr. Inparticular embodiments, N-glycans may be classified into three types:(1) oligomannose, in which only mannose residues are attached to thecore; (2) complex N-glycans, in which “antennae” initiated byN-acetylglucosaminyltransferases (GlcNAcTs) are attached to the core;and (3) hybrid, in which only mannose residues are attached to theManα1-6 arm of the core and one or two antennae are on the Manα1-3 arm.In some embodiments, N-glycans include N-glycans with high mannosecontent, i.e., high mannose N-glycans, bisected and/or sialyl Lewis^(X)N-glycans, or N-acetyl lactosamine containing N-glycans. In someembodiments, examples of various N-linked glycan families include, butare not limited to, (a) the A2 family (disialylated, biantennaryN-linked oligosaccharides; including A1 glycans (monosialylated,biantennary N-linked oligosaccharide), NA2 glycans (asialo-biantennaryN-linked oligosaccharide); NGA2 glycans (asialo-, agalacto-biantennaryN-linked oligosaccharide); M3N2 glycans, etc.); (b) the A2F family(disialylated, biantennary N-linked oligosaccharide with core fucose;including A1F glycans (monosialylated, biantennary N-linkedoligosaccharide with core fucose), NA2F glycans (asialo-biantennaryN-linked oligosaccharide with core fucose) NGA2F glycans (asialo-,agalacto-biantennary N-linked oligosaccharide with core fucose), etc.);(c) the A3 family (e.g., glycans fully sialylated on the non-reducingterminal galactosyl residues but differing in the distribution of a2,3and a2,6 linked sialyl residues and the linkage one of the galactoses;including NA3 (asialo tri-antennary N-linked oligosaccharide derivedfrom an A3 glycan) and NGA3 glycans (agalacto-triantennary N-linkedoligosaccharide derived from an NA3 glycan), etc.); (d) the A4 family(glycans derived from tetra-antennary N-linked oligosaccharides;including NA4 glycans (asialo-tetraantennary N-linked oligosaccharide);NGA4 glycans (asialo-, agalacto-tetraantennary N-linked oligosaccharidederived from NA4 glycans); etc.); and (e) oligomannose family glycans(e.g., Man-5, Man-6, Man-7, Man-8, Man-9, etc. glycans). Examples ofN-glycans include, but are not limited to, a fucosylated biantennarycomplex glycan having no reducing end terminal galactose residues, afucosylated biantennary complex glycan having one reducing end terminalgalactose residue, a fucosylated biantennary complex glycan having tworeducing end terminal galactose residues, a biantennary complex glycanhaving no reducing end terminal galactose residues, a biantennarycomplex glycan having one reducing end terminal galactose residue, abiantennary complex glycan having two reducing end terminal galactoseresidues, a fucosylated biantennary complex glycan having two galactoseresidues and one N-acetylneuraminic acid residue, a fucosylatedbiantennary complex glycan having two galactose residues and twoN-acetylneuraminic acid residues, a biantennary complex glycan havingtwo galactose residues and two N-acetylneuraminic acid residues, a highmannose glycan having five mannose residues, a high mannose glycanhaving six mannose residues, a high mannose glycan having seven mannoseresidues, a high mannose glycan having eight mannose residues, and ahigh mannose glycan having nine mannose residues.

In some embodiments, the agent is any agent that facilitates theremoval, separation, and/or detachment of glycans, e.g., N-glycans, froma glycoconjugate, e.g., a glycoprotein. In certain embodiments, theagent chemically removes the glycan from the glycoconjugate, for examplebut not limited to hydrazinolysis or alkali β-elimination.

In certain embodiments, the agent is an enzyme. In particularembodiments, the agent is an enzyme that specifically removes,separates, and/or detaches N- or O-linked glycans from a glycoconjugate.In certain embodiments, the agent is an amidase. In some embodiments,the agent is or includes a glycosidase, such as an N-glycosidase. Inparticular embodiments, the agent is or includes Endoglycosidase H (EndoH), Endoglycosidase F (EndoF), N-Glycosidase A (PNGase A), orN-Glycosidase F (PNGase F) or combinations thereof. In some embodiments,the agent is or includes an amidase of thepeptide-N4-(N-acetyl-beta-glucosaminyl) asparagine amidase class. Inparticular embodiments the agent is or includes a PNGase F.

In some embodiments, the agent is an enzyme that releases or is capableof releasing full-length oligosaccharides from proteins and peptideshaving N-linked carbohydrates. In some embodiments, the agent is aPNGase F that releases, or is capable of releasing, full-lengtholigosaccharides from proteins and peptides having N-linkedcarbohydrates. In certain embodiments, the agent is not or does notinclude endoglycosidases, such as Endo F, Endo H, and Endo D. In someembodiments, endoglycosidases, such as Endo F, Endo H, and Endo D do notrelease full-length oligosaccharides and/or do not cleave all commonclasses of N-linked oligosaccharides from glycoproteins.

In certain embodiments, the agent is not a protease. In someembodiments, the agent does not include a protease. In particularembodiments, the agent is not serine protease, a cysteine protease, athreonine protease, an aspartic protease, a glutamic protease, ametalloprotease, or asparagine peptide lyases. In certain embodiments,the agent is not and does not include an endopeptidase, e.g., trypsin,chymotrypsin, pepsin, papain, and elastase. In particular embodiments,the agent is not and does not include trypsin.

In particular embodiments, the agent selectively and/or specificallyremoves, separates, and/or detaches an N-glycan from a glycoconjugate,e.g., a surface exposed protein or glycoprotein. In certain embodiments,the agent has a greater activity for the removal, separation, and/ordetachment of an N-glycan than for the removal, separation, and/ordetachment of a glycan that is not an N-glycan. In certain embodiments,the agent has a at least a 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,90%, 100%, or at least a 1-fold, 2-fold, 3-fold, 4-fold, 5-fold,10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold,90-fold, 100-fold, 500-fold, or 1,000 fold greater activity for theremoval, separation, and/or detachment of an N-glycan than for theremoval, separation, and/or detachment of a glycan that is not anN-glycan. In certain embodiments, the agent is or includes a PNGase F.

PNGase F

In particular embodiments, incubation under conditions that are suitableto remove, release, or detach glycans from the surface of cells includescontacting, treating, and/or incubating the cells with an agent. Inparticular embodiments, the agent is or includes a PNGase F. PNGase F isan amidase of the peptide-N4-(N-acetyl-beta-glucosaminyl) asparagineamidase class. In some embodiments, PNGase F is a bacterial enzyme thatreleases N-glycans from an asparagine. In particular embodiments, thePNGase F releases the entire, i.e., intact, N-glycan from theasparagine. In certain embodiments, PNGase F removes oligomannose,hybrid, and complex N-glycans attached to asparagine. In particularembodiments, PNGase F releases N-glycans attached to the nitrogen ofasparagine, thereby converting asparagine to aspartate. In certainembodiments, the cleavage occurs at a position of the carbohydrate thatis adjacent to the asparagine residue. In particular embodiments, theagent is or includes an enzyme that exhibitspeptide-N-(N-acetyl-β-N-glucosaminyl) asparagine aminidase activity. Incertain embodiments, a composition of cells is treated, contacted, orincubated with an agent that is or includes a PNGase F.

In certain embodiments, the agent is or includes a PNGase F polypeptideor a portion thereof. In some embodiments, the agent is a PNGase F thatis derived from a bacteria or purified from an almond emulsion. Inparticular embodiments, the agent is a PNGase F that is derived from abacteria. In some embodiments, the bacteria is Flavobacteriummeningosepticum. In some embodiments, Flavobacterium meningosepticum isalso known as Elizabethkingia meningosepticum. In certain embodiments,the agent contains all or a portion of the amino acid sequence set forthin SEQ ID NO: 1. In particular embodiments, a portion of a PNGase Fpolypeptide is or contains at least 50, at least 60, at least 70, atleast 80, at least 90, at least 100, at least 110, at least 120, atleast 130, at least 140, at least 150, at least 160, at least 170, atleast 180, at least 190, at least 200, at least 210, at least 220, atleast 230, at least 240, at least 250, at least 260, at least 270, atleast 280, at least 290, at least 300, at least 310, at least 320, atleast 330, at least 340, or at least 350 contiguous amino acids of theamino acid sequence set forth in SEQ ID NO: 1. In some embodiments, theagent has at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, or at least 99.5% to all or a portion of the PNGase F aminoacid sequence set forth in SEQ ID NO: 1.

In some embodiments, the PNGase F is or includes a PNGase F preparationof a high purity. In some embodiments, the PNGase F is or includes aPNGase F preparation that is free of proteases. In certain embodiments,the PNGase F is or includes a PNGase F preparation that is free of EndoH, EndoF, and/or PNGase A activity. In particular embodiments, thePNGase F is or includes a PNGase F preparation that is free ofendotoxin. In some embodiments, the PNGase F is or includes a PNGase Fthat is at least 75%, at least 80%, at least 85%, at least 90%, at least95%, at least 96%, at least 97%, at least 98%, at least 99%, at least99.5%, at least 99.7%, at least 99.8%, at least 99.9%, or at least99.99% pure. In particular embodiments, the PNGase F is a preparationwith less than 20%, less than 10%, less than 5%, less than 1%, less than0.5%, less than 0.1%, less than 0.01%, or less than 0.001% non-PNGase Fprotein contaminants. In some embodiments, the PNGase F preparation issubstantially homogenous. In some embodiments, the PNGase F is orincludes a PNGase F that is at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, at least 99.5%, at least 99.7%, at least 99.8%, at least99.9%, or at least 99.99% homogenous.

In some embodiments, the purity and/or homogeneity of the PNGase F is ormay be assessed by any suitable means, including but not limited tosodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE),gel permeation chromatography (GPC), size exclusion chromatography(SEC), liquid chromatography (LC), high performance liquidchromatography (HPLC), mass spectrometry, circular dichroism (SD),nuclear magnetic resonance (NMR), and fluorescence spectroscopy(fluorometry). In some embodiments, purity and/or homogeneity isassessed or determined by SDS-PAGE and protein staining, such as byCoomasie Blue staining.

In certain embodiments, the PNGase F is not derived, obtained, orpurified from an almond emulsion.

In some embodiments, the PNGase F, or a mutant or portion thereof, is arapid-acting PNGase F, such as a PNGase F with activity to cleave orrelease one or more N-glycans in less than 24 hours, such as generallyless than 12 hours, for example, between 15 minutes and 4 hours, such asgenerally no more than 15 minutes, no more than 30 minutes, no more than60 minutes, no more than 2 hours, no more than 3 hours or no more than 4hours.

In some embodiments, PNGase F, or a mutant or portion thereof, isprovided in an amount that is an enzymatically effective amount toaffect release of one or more N-glycans from a glycoprotein orglycoproteins. In some embodiments, the activity is effective forrelease or cleavage of one or more N-glycans from a glycoprotein orglycoproteins that is a native or non-denatured protein, e.g. present inits native structure, such as when purified or when expressed on thesurface of a cell. In particular embodiments, the amount of the PNGase Fis an amount to exhibit the activity (enzymatic activity) to release theone or more N-glycans from the glycoprotein or glycoproteins within acertain period of time and temperature, which is typically a time andtemperature at which a majority of cells of a cell composition remainviable and/or are not detrimentally affected by the incubation. In somecases, the period of time is no more than 12 hours, no more than 8hours, no more than 6 hours, no more than 4 hours, no more than 2 hours,no more than 1 hour, no more than 30 minutes, or any value or range inbetween any of the foregoing. In some embodiments, the temperature isless than or less than about 40° C., such as temperature of about 10°C., about 15° C., about 20° C., about 24° C., about 25° C., about 26°C., about 27° C., about 28° C., about 29° C., about 30° C., about 31°C., about 32° C., about 33° C., about 34° C., about 35° C., about 36°C., about 37° C., about 38° C., about 39° C., or about 40° C., or anyvalue or range in between any of the foregoing. In some embodiments, thetemperature is between or between about 10° C. and 45° C., or between24° C. and 40° C., or between 35° C. and 39° C. In some embodiments, thetemperature is or is about 37° C.

In some embodiments, the enzymatically effective amount results insubstantial deglycosylation of the glycoprotein or glycoproteins duringthe incubation, e.g. for the period of time and at the temperature, suchas activity to effect release or removal of greater than 50%, greaterthan 55%, greater than 60%, greater than 65%, greater than 70%, greaterthan 75%, greater than 80%, greater than 85%, greater than 90%, greaterthan 95%, greater than 99%, about 100%, or 100% of N-glycans present onthe glycoprotein or glycoproteins, including when in native ornon-denatured form, e.g. when present or expressed on the surface of acell.

In some embodiments, the activity of a PNGase F enzyme may be expressedas a unit. In certain embodiments, the units are predefined. Inparticular embodiments, a unit is an amount of PNGase F required toremove an amount of glycan, e.g., N-glycan, from an amount of aglycoprotein, e.g., a purified glycoprotein, in an amount of time underspecific conditions. In some embodiments, the glycoprotein is adenatured glycoprotein. In certain embodiments the glycoprotein is aglycoprotein in its native structure. In some embodiments, the unit isan amount of PNGase F required to remove greater than 50%, greater than55%, greater than 60%, greater than 65%, greater than 70%, greater than75%, greater than 80%, greater than 85%, greater than 90%, greater than95%, greater than 99%, about 100%, or 100% of the glycan from the amountof the glycoprotein. In some embodiments, the reaction volume is about0.1 μl, about 1 μl, about 5 μl, about 10 μl, about 20 μl, about 30 μl,about 40 μl, about 50 μl, about 100 μl, about 200 μl, about 250 μl,about 500 μl, about 1 mL, about 2 mL, about 3 mL, about 4 mL, about 5mL, or about 10 mL, or any value or range in between. In certainembodiments, the amount of the glycoprotein is about 1 pmol, about 10pmol, about 100 pmol, about 1 nmol, about 10 nmol, about 100 nmol, about1 mmol, about 10 mmol, about 100 mmol, about 1 mol, or any value orrange falling in between. In some embodiments, the amount of theglycoprotein is a concentration of about 1 nM, about 10 nM, about 100nM, about 1 μM, about 10 μM, about 100 μM, about 1 mM, about 10 mM,about 100 mM, or about 1 M, or any value or range in between.

In some embodiments, with reference to a unit of activity of a PNGase F,the glycoprotein is a recombinant glycoprotein. In particularembodiments, the glycoprotein is purified. In some embodiments, theglycoprotein is dabsyl fibrin glycopeptide. In certain embodiments, theglycoprotein is fetuin. In particular embodiments, the glycoprotein isdenatured Ribonuclease B (RNase B). In certain embodiments, the unit isfurther defined by conditions of the incubation that include thecomposition of the buffer that the PNGase and the glycoprotein areincubated in, the pH of the buffer, temperature, and the total volume,i.e., reaction volume.

Exemplary units of PNGase F activity include, but are not limited to, aunit as defined as the amount of PNGase F enzyme required to remove >95%of the carbohydrate from 10 μg of denatured RNase B in 1 hour at 37° C.in a total reaction volume of 10 μl; required to catalyze the release ofN-linked oligosaccharides from one nmol of denatured Ribonuclease B in 1minute at pH 7.5 at 37° C.; required to catalyze the deglycosylation of1 nmol of denatured RNase B in 30 minutes at 37° C.; required tocatalyze the release of N-linked oliogosaccharides from 1 μmol denaturedRNase B per minute at pH 7.5 at 37° C.; or required to hydrolyzecarbohydrates from 1 nmol dabsyl fibrin glycopeptide per minute at pH7.8 at 37° C. In particular embodiments, the unit is the amount ofPNGase F activity that is required to catalyze the deglycosylation of 1nmol of denatured RNase B in 30 minutes at 37° C.

Certain embodiments contemplate that one of skill in the art candetermine the amount of PNGase F in a predefined unit as a matter ofroutine. In particular embodiments, the amount of a PNGase F enzyme in aunit is determined by an assay that measures or quantifies the removal,release, deglycosylation, and/or hydroxylation of carbohydrates, e.g.,N-glycans, from a glycoprotein, e.g., a purified glycoprotein. In someembodiments, the PNGase F activity is measured by detecting the amountof N-glycans on a glycoprotein that was treated with PNGase underdefined conditions and comparing it to an amount of N-glycans on anuntreated glycoprotein e.g., a separate pool of untreated glycoproteinor the glycoprotein prior to treatment with the PNGase F.

In some embodiments, methods for detecting N-glycans on the glycosylatedproteins may include staining and affinity-based methods. In certainembodiments, the glycoprotein is run on a gel, e.g., an SDS-PAGE gel,following incubation with the PNGase F enzyme, and optionally stainingthe gel is stained for glycoproteins. In some embodiments, protein bandscorresponding to the glycosylated protein are compared to bandscorresponding to the unglycosylated protein. Suitable gel-stainingprocedures are include, but are not limited to stains that are based onthe periodic acid-Schiff (PAS) reaction, in which periodic acid oxidizestwo vicinal diol groups to form an aldehyde, which reacts with theSchiff reagent to give a magenta color. In some embodiments, thechromogenic gel staining is performed with acid fuchsin, which can bedetected fluorescently at 535 nm. Suitable methods for gel stainingincluding the use of commercially available fluorescent stains thatutilize periodate oxidation to attach a fluorescent hydrazide. Suitablegel stains also include alcian blue and Stains-All, which are used fordetecting proteoglycans, glycosaminoglycans, and negatively chargedglycoproteins. In certain embodiments, the N-glycans by the PGNase Fenzyme is determined by lectin blot, e.g., a western blot that is probedwith a lectin, e.g., a detectably labeled lectin.

In some embodiments, the enzymatically effective amount of theN-glycosidase, e.g. PNGase F, is from or from about 1 unit to 5000units, 1 unit to 1000 units, 1 unit to 500 units, 1 unit to 250 units, 1unit to 100 units, 1 unit to 50 units, 1 unit to 25 units, 25 units to5000 units, 25 units to 1000 units, 25 units to 500 units, 25 units to250 units, 25 units to 100 units, 25 units to 50 units, 50 units to 5000units, 50 units to 1000 units, 50 units to 500 units, 50 units to 250units, 50 units to 100 units, 100 units to 5000 units, 100 units to 1000units, 100 units to 500 units, 100 units to 250 units, 250 units to 5000units, 250 units to 1000 units, 250 units to 500 units, 500 units to5000 units, 500 units to 1000 units, or 1000 units to 5000 units, eachinclusive. In some embodiments, the enzymatically effective amount ofthe N-glycosidase, e.g. PNGase F, is greater than or greater than aboutor is or is about 1 unit, 5 units, 10 units, 15 units, 20 units, 25units, 50 units, 100 units, 250 units, 500 units, 1000 units, 2500 unitsor 5000 units. It is within the level of a skilled artisan to determinethe unit of activity and/or specific activity of a preparation of anN-glycosidase, e.g. PNGase F. Exemplary methods for assessing ordetermining unit of activity are described above, and may depend on theparticular source of the N-glycosidase, e.g. PNGase F. In some instance,one unit is an amount of the N-glycosidase, optionally PNGase F,sufficient to catalyze the deglycosolation of 1 nanomole of denaturedRibonuclease B (RNase B) in 30 minutes at 37° C. In other instances, 500units is an amount of the N-glycosidase, optionally PNGase F, sufficientto catalyze the deglycosylation of 10 μg of Ribonuclease B (RNase B)incubated in 1XPBS for 5-10 minutes at 37° C. or room temperature.

In some embodiments, the PNGase F is a recombinant PGNase F. In certainembodiments, the PNGase F a mutant PNGase F. In some embodiments, thePNGase F is a recombinant PNGase F that is cloned from Flavobacteriummeningosepticum. In particular embodiments, the PNGase F is cloned fromthe entire PNGase F gene of Flavobacterium meningosepticum. In certainembodiments, the entire PNGase F gene of Flavobacterium meningosepticumis the PNGase F gene described in Tarentino et al., Journal ofBiological Chemistry, 265(12): 6961-6966 (1990). In particularembodiments, the entire PNGase F gene is a PNGase F gene that encodes aPNGase F polypeptide that is designated with the Uniprot Accessionnumber P21163.2. In some embodiments, the entire PNGase F gene is aPNGase F gene that encodes a PNGase F polypeptide with the amino acidsequence set forth in SEQ ID NO: 1.

In particular embodiments, the PNGase F achieves completedeglycosylation of RNase B within 5-10 minutes at 37° C. or at roomtemperature. In some embodiments, the PNGase F with an increased and/orrapid activity is a PNGase F that achieves complete deglycosylation of10 μg of RNase B when one unit of the PGNase F is incubated for 5-10minutes at 37° C. or at room temperature. In particular embodiments, thecomplete deglycosylation is visualized by SDS-PAGE. In particularembodiments, the purity of the PNGase F at least 95% or greater asdetermined by SDS-PAGE analysis and staining with Coomassie BrilliantBlue. In some embodiments, the PNGase F releases N-glycans from thenative form of the blood borne glycoprotein fetuin within minutes atroom temperature or at 37° C.

In some embodiments, the PNGase F is a PNGase F that is capable ofremoving, releasing, and/or detaching N-glycans within minutes whenincubated with a native glycoprotein at room temperature or at 37° C. Inparticular embodiments, the PNGase F is capable of removing, releasing,and/or detaching an amount of N-glycans from frozen and formalin-fixedparaffin-embedded liver tissue sections within 2 hours at 37° C.,wherein the amount of released N-glycans is detectable by massspectrometry, e.g., MALDI-MS (see for example Powers et al. AnalyticalChemistry, 85(20):9799-806 (2013). In certain embodiments, the PNGase Fis capable of removing, releasing, and/or detaching an amount ofN-glycans from frozen mouse brain tissue sections when 0.2 mL solutionof 0.1 mg/mL is applied to the section and incubated for 2 hours at 37°C., wherein the amount of released N-glycans is detectable by massspectrometry, e.g., MALDI-MS (see for example, Powers et al.,Biomolecules 5: 2554-2572 (2015)).

In particular embodiments, the PNGase F deglycosylates proteins in theirnative form. In certain embodiments, the PNGase F removes N-glycans fromone or more glycoproteins that are in their native form. In someembodiments, the PNGase F releases, removes, and/or detaches glycansfrom one or more glycoproteins that are in their native form withinminutes at room temperature. In certain embodiments, the PNGase Freleases, removes, and/or detaches glycans from one or moreglycoproteins that are in their native form within minutes at roomtemperature or at 37° C. In certain embodiments, the PNGase F releases,removes, and/or detaches at least 10%, at least 20%, at least 30% atleast 40%, at least 50%, at least 60%, at least 70%, at least 80%, atleast 90%, at least 95%, at least 99%, or about 100% of glycans from oneor more glycoproteins in their native form within about 5 minutes, about10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about30 minutes, about 35 minutes, about 40 minutes, about 45 minutes, about50 minutes, about 55 minutes, about 60 minutes, about 1.5 hours, about 2hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, orbetween 5 minutes and 1 hour, between 1 hour and 4 hours, between 4hours and 6 hours, or longer than 6 hours.

In particular embodiments, the PNGase F is a recombinant PNGase F thatis produced by a polynucleotide that is cloned from a PNGase F gene thatis expressed and purified in cells. In some embodiments, the cells arebacterial cells, e.g., E. coli, yeast cells, insect cells, or mammaliancells. In certain embodiments, the PNGase F is expressed in and purifiedfrom E. coli. In some embodiments, the PNGase F is expressed in andpurified from the bacterial strain BL21 Star (DE3).

In some embodiments, the PNGase F is a recombinant PNGase F that isproduced by a polynucleotide that is cloned, expressed, and purifiedinto an expression vector. In certain embodiments, the polynucleotideencoding the PNGase F is incorporated into a vector construct containinga regulatory sequence by routine molecular techniques (See Sambrook etal, Molecular Cloning, 2nd ed., (1989)). In some embodiments, the vectorincludes one or more of a suitable promoter, origin of replication,ribosomal binding site, transcription termination sequence, selectablemarkers and multiple cloning sites. In particular embodiments, thepolynucleotide encoding the PNGase F is a plasmid with an efficient andspecific construct, e.g., a T7 expression vector. In certainembodiments, the vector contains an inducible promoter. In someembodiments, the polynucleotide encoding the PNGase F is inserted intoan expression vector that is suitable for expression in a bacteriumunder the control of a suitable promoter for bacteria. In particularembodiments, the expression vector contains an inducible promoter thatis recognized by the host bacterial organism and is operably linked tothe polynucleotide encoding the PGNase F. Inducible promoters suitablefor use with bacterial hosts include the β-lactamase and lactosepromoter systems, the arabinose promoter system, including the araBADpromoter, the rhamnose promoter, an alkaline phosphatase promoter, atryptophan (trp) promoter system, a PLtet0-1 and Plac/ara-1 promoters,and hybrid promoters such as the tac promoter. In some embodiments,other known bacterial inducible promoters and low-basal-expressionpromoters are suitable. In certain embodiments, the expression vectorcontains a lacUV5 promoter and allows for highlevelisopropyl-beta-D-thiogalactopyranoside (IPTG) inducible expressionof gene products from T7 expression vectors such as pET and pQE.

In some embodiments, producing the PGNase F a process whereby thepolynucleotide encoding the PNGase F is cloned, expressed, and purifiedinto a T7 expression vector. T7 expression vectors include, but are notlimited to, commercially available T7 vectors such as pT7 FLAG 3, pT7FLAG 1, pT7 MAT 1, pT7 FLAG 4, pT7 FLAG 2, pT7 MAT 2, pT7 FLAG-MAT 1,pT7 MAT-FLAG 2, pT7 MAT-FLAG 1, and pT7 FLAG-MAT 2 (Sigma), GATEWAYpDEST 14, GATEWAY pDEST 15, GATEWAY pDEST 16, GATEWAY pDEST 17, pRSET A,pRSET-BFP, pRSET-CFP, pRSET-EmGFP (Thermo Fisher), pET 29-b (Novagen)and pQE-T7(Qiagen). In certain embodiments, the polynucleotide encodingthe PNGase F is cloned, expressed and purified into a pET 29-b and/or apQE-T7T7 vector.

In some embodiments, the PNGase F contains a tag or fusion domain, e.g.affinity or purification tag, linked, directly or indirectly, to the N-and/or c-terminus of the protein. In particular embodiments, the PGNaseF is produced by a process whereby the PNGase F is a recombinant PNGaseF that is cloned, expressed and purified into an expression vector. Insome embodiments, the insertion results in the addition of the sequencefor an in frame N-terminal or C-terminal tag and/or a fusion domain. Insome embodiments, the tag and/or fusion domain is an N-terminal tag ofthe PNGase F polypeptide. In particular embodiments, the tag and/orfusion domain is a C-terminal tag of the PNGase F polypeptide. Varioussuitable polypeptide tags and/or fusion domains are known, and includebut are not limited to, a FITC tag, poly-histidine (His), HRP, maltosebinding protein, Glu-Glu, avidin, glutathione S transferase (GST),protein A, protein G, an immunoglobulin heavy chain constant region(Fc), human serum albumin, AviTag, a Calmodulin-tag, a polyglutamatetag, a FLAG-tag, an HA-tag, a Myc-tag, and fluorescent protein-tags(e.g., EGFP). In particular embodiments, the PNGase F a recombinantPNGase F with a C-terminal His tag.

In certain embodiments, the PGNase F is produced by a process wherebybacterial cells transduced with the vector containing the polynucleotidethat encodes the PNGase F are induced to express the PNGase F protein.In particular embodiments, bacterial cells expressing the PNGase Fpolypeptide are harvested and lysed, and the PNGaseF polypeptide ispurified. In some embodiments, the PNGase F polypeptide is purified.Suitable techniques for use in protein purification include, but are notlimited to, precipitation with ammonium sulfate, PEG, antibodies and thelike, or by heat denaturation, followed by: centrifugation;chromatography steps such as ion exchange, gel filtration, reversephase, hydroxylapatite and affinity chromatography; isoelectricfocusing; gel electrophoresis; and combinations of these and othertechniques. In some embodiments, the purification of the PNGase Fpolypeptide is performed by HPLC or FPLC purification. In someembodiments, the protein is purified by methods whereby a column, bindsto or retains the PGNase F polypeptide by interacting with a proteintag, e.g., a C-terminal his tag, of the recombinant PGNase Fpolypeptide.

In particular embodiments, the agent is or includes the PNGase F that isproduced from a polynucleotide that is cloned from the entire PNGase isentire Peptide N-Glycosidase F(PNGase F) gene from the genome ofFlavobacterium meningosepticum, expressed and purified into the T7expression vectors pET 29-b (Novagen) and pQE-T7(Qiagen). In certainembodiments, the polynucleotide that encodes the PNGase F contains anin-frame C-terminal histidine tag. In some embodiments, thepolynucleotide encoding the PNGase F HIS-tagged construct is transformedinto bacterial strain BL21 Star (DE3) that carries the gene for the T7RNA polymerase under control of the lacUV5 promoter which allows forhigh level isopropyl-beta-D-thiogalactopyranoside (IPTG) inducibleexpression of gene products from T7 expression vectors such as pET andpQE. In particular embodiments, bacterial transformation and cellculture growth is performed, bacterial cells are harvested bycentrifugation, and cell pellets are washed with buffers containingprotease inhibitors (SigmaFast EDTA-free). In particular embodiments,the total cellular protein lysates are made using an Avestin C5 highpressure homogenizer. In some embodiments, FPLC purification methods forthe recombinant PNGase F histidine tagged protein use Ni-NTA (Qiagen)and IMAC HisTrap HP (GE Healthcare) columns. In some embodiments,bacterial cell lysate from IPTG induced cultures are loaded onto thecolumn and bound the PNGase F polypeptide with the C-terminal His tag iswashed and eluted using an imidazole step gradient in binding buffer. Insome embodiments, purified PNGase F with the C-terminal is dialyzed andstored in PBS buffer. In particular embodiments, the agent is orincludes a PNGase that is a recombinant PNGase F with a C-terminal Histag or is a PNGase F that is identical to a PNGase F produced by themethods described in Powers et al. Analytical Chemistry, 85(20):9799-806(2013).

In some embodiments, the agent is or includes a PNGase F that is acommercially available PNGase F. Commercially available PNGase Fincludes, but is not limited to, PNGase F Proteomics Grade (Catalog # P7367, Sigma); PNGase F (Catalog # P0704S and P0704L, New EnglandBiolabs), PNGase F (Catalog # V4831, Promega), N-GLYCANASE (Catalog #:GKE-5006A, GKE-5006B, GKE-5006D, GKE-5016A, GKE-5016B, GKE-5016D,GKE-5010B, GKE-5016D, GKE-5020B, GKE-5020D, and GKE-5003, ProZyme), andPNGase F (Catalog #: E-PNG01, QA Bio),RAPID PNGase F (Catalog # P0710S,New England Biolabs), PNGASE F PRIME (N-Zyme Scientifics). In certainembodiments, the PNGase F is or is identical to PNGASE F PRIME (N-ZymeScientifics).

2. Incubation Conditions

In some embodiments, a composition of cells, e.g. test composition, iscontacted, treated, and/or incubated with an agent, such as anN-glycosidase, e.g. PNGase F, for about 5 minutes, about 10 minutes,about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes,about 35 minutes, about 40 minutes, about 45 minutes, about 50 minutes,about 55 minutes, about 60 minutes, about 1.5 hours, about 2 hours,about 3 hours, about 4 hours, about 5 hours, about 6 hours, or between 5minutes and 1 hour, between 1 hour and 4 hours, between 4 hours and 6hours, or longer than 6 hours. In particular embodiments, thecomposition of cells is contacted, treated, and/or incubated with anagent that is or includes a PGNase F for an amount of time between 30minutes and 60 minutes. In certain embodiments, a composition of cellsis contacted, treated, or incubated with an agent that is or includes aPNGase F for about 30 minutes. In certain embodiments, a composition ofcells is contacted, treated, or incubated with an agent that is orincludes a PNGase F for about 60 minutes.

In particular embodiments, a composition of cells, e.g. test cellcomposition, is contacted, treated, or incubated with an agent, such asan N-glycosidase, e.g. PNGase F, and at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, at least 97%, atleast 98%, at least 99% or at least 99.9% of the surface exposedN-glycans are removed from the cells in an amount of time that is lessthan 6 hours, less than 5 hours, less than 4 hours, less than 3 hours,less than two hours, less than 90 minutes, less than 60 minutes, lessthan 45 minutes, less than 30 minutes, less than 15 minutes, or lessthan 5 minutes. In certain embodiments, the composition of cells, e.g.test cell composition, is incubated with an agent, such as anN-glycosidase, e.g. PNGase F, for about 5 minutes, about 10 minutes,about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes,about 35 minutes, about 40 minutes, about 45 minutes, about 50 minutes,about 55 minutes, or about 60 minutes or between 5 minutes and 60minutes, between 5 minutes and 30 minutes, between 30 minutes and 60minutes, or between 15 minutes and 45 minutes and at least 85%, at least90%, at least 95%, or at least 99% of the surface exposed N-glycans areremoved from the cells. In particular embodiments, the composition ofcells, e.g. test cell composition is incubated with an agent, such as anN-glycosidase, e.g. PNGase F, for about 30 minutes and at least 85%, atleast 90%, at least 95%, or at least 99% of the surface exposedN-glycans are removed from the cells. In certain embodiments, thecomposition of cells, e.g. test cell composition, is incubated with anagent, such as an N-glycosidase, e.g. PNGase F for about 60 minutes andat least 85%, at least 90%, at least 95%, or at least 99% of the surfaceexposed N-glycans are removed from the cells.

In some embodiments, a composition of cells, e.g. test cell composition,is incubated with an agent, such as an N-glycosidase, e.g. PNGase F, toremove N-glycans from the surface of the cells under conditions that donot damage or kill the cells. In some embodiments, the incubation is forno more than or about 5 minutes, no more than or about 10 minutes, nomore than or about 15 minutes, no more than or about 20 minutes, no morethan or about 25 minutes, no more than or about 30 minutes, no more thanor about 35 minutes, no more than or about 40 minutes, no more than orabout 45 minutes, no more than or about 50 minutes, no more than orabout 55 minutes, no more than or about 60 minutes, no more than orabout 1.5 hours, no more than or about 2 hours, no more than or about 3hours, no more than or about 4 hours, no more than or about 5 hours, orno more than or about six hours, wherein less than about 20%, about 15%,about 10%, about 5%, about 1%, about 0.5%, about 0.1%, about 0.05%,about 0.01%, about 0.001%, or about 0.0001% of the cells in thecomposition die, rupture, lyse, and/or initiate or undergo apoptosis ornecrosis during the incubation. In some embodiments, the incubation isfor a time that is no more than or is about 30 minutes or 60 minutes, oris an amount of time between 30 and 60 minutes, inclusive, and less thanabout 20%, about 15%, about 10%, about 5%, about 1%, about 0.5%, about0.1%, about 0.05%, about 0.01%, about 0.001%, or about 0.0001% of thecells in the composition die, rupture, lyse, and/or initiate or undergoapoptosis or necrosis during the incubation.

In certain embodiments, the contact, treatment, and/or incubation withthe agent, e.g., N-glycosidase, is performed with a rocking motion. Incertain embodiments, the rocking is, is about, or is at least 25 RPM, 50RPM, 100 RPM, 150 RPM, 200 RPM, 250 RPM, 300 RPM, 350 RPM, 400 RPM, 450RPM, or 500 RPM. In some embodiments, the rocking is or is about 250RPM. In some embodiments, the contact, treatment, and/or incubation withthe agent, e.g., N-glycosidase, is performed under stationaryconditions. In some embodiments, the cells are briefly mixed and/orvortexed e.g., for, for about, or for less than 30, 15, 10, 5, 2, or 1seconds, during the contact, treatment, and/or incubation with theagent. In some embodiments, the cells are mixed or vortexed once, twice,three times, four times, five times, six times, seven times, eighttimes, nine times, ten times, or more than ten times during thecontacting, incubation, and/or treatment.

In some embodiments, the composition of cells, e.g. test cellcomposition, is contacted, treated, and/or incubated with the agent,such as N-glycosidase, e.g. PNGase F, at a temperature less than 10° C.In some embodiments, the temperature is between 10° C. and 45° C., orbetween 24° C. and 40° C., or between 35° C. and 39° C. In someembodiments, the temperature is at least or at least about or is or isabout 10° C., 15° C., 20° C., 24° C., 25° C., 26° C., 27° C., 28° C.,about 29° C., 30° C., 31° C., 32° C., 33° C., 34° C., 35° C., 36° C.,37° C., 38° C., 39° C., or 40° C., or any range between any of theforegoing. In certain embodiments, a composition of cells is contacted,treated, or incubated with an agent that is or includes a PNGase F at atemperature of about 37° C.

In particular embodiments, a composition of cells is contacted, treated,or incubated with an agent, such as an N-glycosidase, e.g. PNGase F, andat least 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95%, at least 97%, at least 98%, at least 99% or at least 99.9% ofthe surface exposed N-glycans are removed from the cells after anincubation performed at a temperature between 10° C. and 45° C., orbetween 24° C. and 40° C., or between 35° C. and 39° C., such as atleast or at least about or is or is about 10° C., 15° C., 20° C., 24°C., 25° C., 26° C., 27° C., 28° C., about 29° C., 30° C., 31° C., 32°C., 33° C., 34° C., 35° C., 36° C., 37° C., 38° C., 39° C., or 40° C.,or any range between any of the foregoing In certain embodiments, thecomposition of cells is incubated with an agent that is or includes aPNGase F at a temperature of between 10° C. and 45° C., or between 24°C. and 40° C., or between 35° C. and 39° C., such as at least or atleast about or is or is about 10° C., 15° C., 20° C., 24° C., 25° C.,26° C., 27° C., 28° C., about 29° C., 30° C., 31° C., 32° C., 33° C.,34° C., 35° C., 36° C., 37° C., 38° C., 39° C., or 40° C. or any rangebetween any of the foregoing, and at least 85%, at least 90%, at least95%, or at least 99% of the surface exposed N-glycans are removed fromthe cells.

In some embodiments, a composition of cells is incubated with an agent,such as an N-glycosidase, e.g. PNGase F to remove N-glycans from thesurface of the cells under conditions that do not damage or kill thecells. In some embodiments, the incubation is at a temperature ofbetween 10° C. and 45° C., or between 24° C. and 40° C., or between 35°C. and 39° C., such as at least or at least about or is or is about 10°C., 15° C., 20° C., 24° C., 25° C., 26° C., 27° C., 28° C., about 29°C., 30° C., 31° C., 32° C., 33° C., 34° C., 35° C., 36° C., 37° C., 38°C., 39° C., or 40° C. or any range between any of the foregoing, andless than about 20%, about 15%, about 10%, about 5%, about 1%, about0.5%, about 0.1%, about 0.05%, about 0.01%, about 0.001%, or about0.0001% of the cells in the composition die, and/or initiate or undergoapoptosis or necrosis during the incubation.

In some embodiments, a composition of cells is incubated, treated, andor contacted with a PNGase F and surface expressed N-glycans arereleased, removed, or detached from the surface of the cells. In certainembodiments, the cell composition is incubated at or at about 37° C. forabout 30 minutes, for about 60 minutes, or for an amount of time between30 minutes and 60 minutes. In some embodiments, the incubation isperformed with rocking at or at about 250 RPM. In some embodiments, thePNGase F contains a fusion domain. In certain embodiment, the fusiondomain is a C-terminal His tag. In particular embodiments, the PNGase Fis a preparation that is at least 95% pure and/or is a preparation withless than 5% non-PNGase F protein contaminants. In some embodiments, thePNGase F exhibits deglycosylation activity to release N-glycans from anative or non-denatured protein after incubation for 30-60 minutes at atemperature of 37°±2° C. In some embodiments, a composition of between1×10⁶ cells and 5×10⁶ cells or between 1×10⁶ cells and 2.5×10⁶ cells istreated, contacted, or incubated with an agent that is or includes aPNGase F.

3. Removal of Cells

In some embodiments, the conditions, including incubating the cells withan agent that removes, released, and or detaches glycans, e.g.,N-glycans from proteins expressed on the surface of the cells, resultsin release of glycans into the solution or the media where thetreatment, incubation, and/or the contacting is performed. In certainembodiments, the cells of the composition are intact after theincubation, e.g., the cells are not ruptured, lysed, dying, and/or dead.In particular embodiments, the cells are removed from the media orsolution after the incubation is performed. In particular embodiments,the cells are removed from the media or solution in a manner that doesnot rupture, lyse, and/or kill the cells. In some embodiments cells areremoved by centrifugation, e.g., a low speed and/or low gcentrifugation, and the supernatant is removed from the pellet thatcontains the cells. In particular embodiments, the supernatant contains,the glycans, e.g., N-glycans, that were removed from the surface of thecells during the treatment, incubation, or contacting.

In particular embodiments, the cells are removed from a sample,solution, or media that contains released surface glycans. In certainembodiments, the glycans are removed and/or separated from the media orsolution. In some embodiments, the solution or media is evaporated. Inparticular embodiments, the solution or media is evaporated by vacuumcentrifugation, e.g., with a speedvac. In particular embodiments, theglycans are removed and/or separated from the media or solution and arethen resuspended. In some embodiments, the glycans may be resuspended ina volume of a buffer or solution. In some embodiments, the buffer orsolution is suitable for storage. In certain embodiments, the buffer issuitable for use with a technique for the detection, identification,and/or detection of the glycans. In certain embodiments, the buffer orsolution is suitable for a chemical reaction, e.g., a derivationreaction such as the addition of a detectable label.

B. Detecting Glycans

In some embodiments, after the incubation in accord with the providedmethods, the glycans, e.g. N-glycans, that are attached to proteinsexpressed and/or exposed on the surface of the cells are released,removed, and/or detached into a media and or a solution. In certainembodiments, at least one, at least two, at least three, at least four,at least five, at least six, at least seven, at least eight, at leastnine, at least ten, at least eleven, at least twelve, at least thirteen,at least fourteen, at least fifteen, at least sixteen, at leastseventeen, at least eighteen, at least nineteen, at least twenty, atleast twenty-five, at least thirty, at least thirty-five, at leastforty, at least forty-five, at least fifty, at least fifty-five, atleast sixty, at least sixty-five, at least seventy, at leastseventy-five, at least eighty, at least eighty-five, at least ninety, atleast ninety-five, at least 100, at least 125, at least 150, at least175, at least 200, at least 225, at least 250, at least 300, at least350, at least 400, at least 450, or at least 500 or greater species ofglycans, e.g. N-glycans, are into the media or solution. In particularembodiments, the species of glycans, e.g. N-glycans, are detectablespecies of glycan. In some embodiments, the detectable species areN-glycans that can be detected, identified, measured, and/or quantifiedwithin the sample or from a preparation that is produced, generated,prepared, and/or derived from the sample.

Provided herein are methods for generating a high resolution surfaceN-glycan map of a cell composition. In some embodiments, the methodsinclude the steps of detecting N-glycans that have been removed,released, and/or detached from the cell surface, e.g., fromglycoproteins exposed at the cell surface. In particular embodiments,the detection of the N-glycans includes identifying and/or quantifyingthe N-glycans, for example by mass spectrometry (MS) or high performanceliquid chromatography (HPLC) to produce the high resolution surfaceN-glycan expression profile. In certain embodiments, the methodsprovided herein include steps for washing, cleaning, and or purifying asample of released N-glycans. In certain embodiments, the methodsprovided herein include steps for derivatizing the N-glycans, forexample to increase or enhance detection of the N-glycans.

1. Derivation and Purification

In certain embodiments, glycans, e.g. N-glycans, are modified to improveand/or enhance the detection of the glycans. In many instances, glycansmay not be readily detectable due to the absence of a strong chromophoreor fluorophore or active moiety that is detectable by liquidchromatography and/or mass spectrometry. In some embodiments, theabsorbance and fluorescence response of a glycan may be relatively weakor below a threshold for detection. In some embodiments, one tactic tomaximize the sensitivity of an assay is to convert the compound ofinterest, i.e., the glycan, into a derivative that exhibits a betterresponse for the particular detection method being utilized. In certainembodiments, the derivatizing agent affects or influences the ultimatesensitivity and accuracy of an analysis by maximizing the sensitivity,yield and/or stability of the derivatized molecules. Thus, in someembodiments, the glycans (e.g., N-glycans) that have been released fromcellular surfaces are derivatized prior to any procedures for analysisor detection.

In some embodiments, the glycans are derivatized prior to an analysis byHPLC and/or mass spectrometry. In some embodiments, the sensitivity ofthe detection of N-glycans by existing techniques, e.g., highperformance liquid chromatography (HPLC) and/or optical or massspectrometric (MS) detection, can be improved and/or enhanced by aderivation step.

In some embodiments, a glycan, e.g., an N-glycan, is derivatized toallow for or improve detection by mass spectrometry. In certainembodiments, the glycan is derivatized to allow for the glycan to moreeasily accept a charge. In certain embodiments, a glycan and/or aderivatized glycan that is capable of accepting charge is detectable bya mass spectrometer. In some embodiments, the glycan is derivatized byadding an amino group, e.g., a tertiary amino group.

In some embodiments, the derivatization is or includes adding adetectable label to the glycans, e.g., N-glycans. In some embodiments,the addition is a covalent attachment. In certain embodiments, theattached detectable label increases signal and/or reduce backgroundnoise during the detection of the N-glycans as compared to detection ofN-glycans that do not contain an attached detectable label. In certainembodiments, any of a variety of detectable labels can be used inaccordance with the present disclosure, including but not limited to,fluorescent labels, radiolabels and/or chemiluminescent labels. Incertain embodiments, the detectable label is a fluorescence label. Incertain embodiments, attachment, e.g., covalent attachment, of thefluorescence label does not alter migration of the N-glycan in a column,e.g., a column suitable for HPLC. In particular embodiments, the labelis a fluorescence label and allows for the glycan to more easily accepta charge as compared to an unlabeled glycan.

In some embodiments, released glycans can be analyzed by massspectrometry, e.g., MALDI MS or ESI-MS-MS, directly without anyderivation and/or chemical tagging. In some embodiments, this label-freeapproach is suitable for qualitative analysis for glycans. However, insome embodiments, the label-free approach is not as well suited forrelative quantitation due to the fact that glycans from a single proteinsample can be very heterogeneous in that the ionization efficiency isnot the same among them. Therefore, in some embodiments, a singleanalysis platform that can perform both quantitative and qualitativeanalysis is used, for example, to determine the profile of surfaceN-glycan expression. A fluorescent detector only detects the dye itself,the so fluorescent response from various glycans can be used forrelative quantitation. In some embodiments, a derivatizing or labelingreagent is used for an analytical procedure.

In some embodiments, the derivatization of the N-glycans is performed bya standard technique in the art. A large number of N-glycanderivatization techniques have been described and are reviewed in Ruhaaket al., Analytical and Bioanalytical Chemistry 397(8): 3457-3481 (2010).In some embodiments, the derivatization is performed by a chemicalreaction that includes two or more reaction steps. In some embodiments,derivatization is performed by reaction reductive amination,permethylation, Michael addition, or hydrazide labeling. In certainembodiments, various compounds which provide the required functionalgroup for the labeling reaction can be used. In certain embodiments, thederivatization is performed by a chemical reaction with a singlereaction step. Labeling agents that add a label to a glycan by Chemicalreaction with a single reaction step that are suitable forderivatization and/or covalently attaching a detectable label to anN-glycan includes agents that contain a functional group that rapidlyreacts with amines (such as an isocyanate, or succidimidylcarbamate).Such labeling agents and fluorescence labels are described in U.S. Pat.App. No: US 20140242709.

In certain embodiments, the N-glycans are labeled by reductiveamination. In this reaction, a label containing a primary amine groupreacts in a condensation reaction with the aldehyde group of the glycan,resulting in an imine or Schiff base, which is reduced by a reducingagent to yield a secondary amine. In some embodiments, the reaction isperformed in dimethyl sulfoxide containing acetic acid, tetrahydrofuran,or methanol. In some embodiments, reductive amination results in thestoichiometric attachment of one label per N-glycan allowing a directquantitation based on fluorescence or UV-absorbance intensity.

Various labels have been used for the reductive amination of glycans. Insome embodiments, fluorescent label that is or includes 2-aminobenzamide(2-AB), 2-aminobenzoic acid (2-AA), 2-aminopyridine (PA),2-Aminoacridone (AMAC), 2-aminonaphthalene trisulfonic acid (ANTS), and1-aminopyrene-3,6,8-trisulfonic acid (APTS),3-(Acetylamino)-6-aminoacridin (AA-Ac), 6-Aminoquinoline (6-AQ),7-Aminomethyl-coumarin (AMC), 2-Amino (6-amido-biotinyl) pyridine (BAP),9-Fluorenylmethoxycarbonyl (FMOC)-hydrazide,1,2-Diamino-4,5-methylenedioxy-benzene (DMB), or o-Phenylenediamine(OPD) is added to the glycans.

In particular embodiments, the N-glycans are labeled with a commerciallyavailable label. Labeling kits are available for the tags 2-AB, 2-AA,and PA (Ludger) as well as for labeling with APTS (Beckmancoulter) andANTS (Prozyme). In some embodiments, the labeling agent and/or thefluorescence label is RapiFluor-MS (Waters Technologies Corporation).

In some embodiments, the labeling agent contains a fluorescent moiety,and a functional group that rapidly reacts with amines (such as anisocyanate, or succidimidylcarbamate). In some embodiments, the labelingagent contains one or more of a tertiary amino group or other MS activeatom, a fluorescent moiety, and a functional group that rapidly reactswith amines (such as an isocyanate, or succidimidylcarbamate).

In particular embodiments, the labeling agent contains a fluorescentmoiety that is or includes phanquinones or benzooxadiazoles. In someembodiments, the labeling reagent contains a fluorescent moiety that isa coumarin. Suitable examples of coumarins include, but are not limitedto, coumarin 7 (3-(2,-Benzimidazolyl)-7-N,N-diethylaminocoumariii), NileRed derivative, Coumarin 4 (7-Hydroxy-4-metbylcoumarin); Coumarin 120(7-Amino-4-methyicoumarin); Coumarin 2 (7-Amino-4 methyl coumarin);Coumarin 466 (7-Diethylaminocoumarin); Coumarin 47(7-Diethylamino-4-methylcoumarin); Coumarin 6H(2,3,5,6-1H,4H-Tetrahydroquinolizino-[9,9a, 1-gh]coumarin); Coumarin152A (7-Diethylamino-4-trifluormethylcoumarin); Coumarin 152(7-Diniethyiamino-4-trifiuomiethylcoumarin); Coumarin 151(7-Amino-4-ti-ifiuormethylcoumarin); Coumarin 6H(2,3,5,6-1H,4H-Tetrahydroquinolizino-[9, 9a, 1-gh]coumarin); Coumarin307 (7-Ethylamino-6-methyl-4-trifluormethylcoumarin); Coumarin 500(7-Ethylamino-4-trifluormethylcoumarin); Coumarin 314(2,3,5,6-1H,4H-Tetrahydro-9-carboethoxyquinolizino-[9,9a,1-gh]coumarin); Coumarin 510(2,3,5,6-U-4H-Tetrahydro-9-(3-pyridyi)-quinolizino-[9,9a, gh] coumarin);Coumarin 30 (3-2′-N-Methylbenzimidazolyl)-7-N,N-diethylaminocoumarin);Coumarin 552 (N-Methyl-4-trifiuormethylpiperidmo-3,2-g]-coumarin);Coumarin 6 (3-(2′-Benzothiazolyl)-7-diethylaminocoumarin).

In some embodiments, the labeling agent contains a fluorescent moietythat is a rhodamine. Suitable rhodamines include, but are not limitedto: Rhodamine 110 (o-(6-Amino-3-imino-3H-xanthen-9-yl)-benzoic acid);Rhodamine 19 (Benzoic Acid,2-[6-(ethylamino)-3-(ethylimino)-2,7-dimethyl-3H-xanthen-9-yl].perchlorate);Rhodamine 6G (BenzoicAcid,2-[6-(ethylamino)-3-(ethylimino)-2,7-dimethyl-3H-xanthen-9-yl]-ethylester,monohydrochloride);Rhodamine B(2-[6-(Diethylamino)-3-(diethylimino)-3H-xanthen-9-yl]benzoic acid). Insome embodiments, the fluorescent moiety is a fluorescein. Fluorsceinsmay include, but are not limited to, Uranin (Disodium Fluorescein); andFluorescein 27 (2,7-Dichloro fluorescein).

In certain embodiments, the labeling agent includes a fluorescent moietythat is a phenyl-substituted oxazol or a furan. In some embodiments, thefluorescent moiety is a PPO (2,5-Diphenyloxazoi); alpha-NPO(2-(1-Naphthyl)-5-phenyloxazol); BBO (2,5-Bis-(4-biphenylyl)-oxazol);and POPOP (1,4-Di[2-(5-phenyloxazolyl)]benzene). In certain embodiments,the fluorescent moieties include quaterphenyls. In particularembodiments, quaterphenyls include TMQ(3,3′,2′,3′″-Tetramethyl-p-quaterphenyl); BMQ(2,2′″-Dimethyl-p-quaterphenyl); DMQ(2-Methyl-5-t-butyi-p-quaterphenyl); PQP (p-Quaterphenyl); Polyphenyl 1(p-Quaterphenyl-4-4′″-disulfonic acid Disodium salt); Polyphenyl 2(p-Quaterphenyl-4-4″-disuifonicacid Dipotassium. salt; BiBuQ(4,4′″-Bis-(2-butyloctyloxy)--quaterphenyl); BM-Terphenyl(2,2″-Dimethyl-p-terphenyl); and FTP (p-Terphenyl). In some embodiments,the fluorescent moiety is a azaquinolone or carbostyryl, including butnot limited to Carbostyryl 7 (7-Ammo-4-methylcarbostyryl); Carbostyryl 3(7-Dimethylamino-4-methylquinolon-2); and Quinolon 390(7-Dimethylamino-1-methyl-4-methoxy-8-azaqumolone-2). In someembodiments, the fluorescent moiety is a benzoxazole, benofurans, orbenzothiazoles, where examples include: DASBTI(2-(p˜Dimethylamiiiostyrj)-benzothiazolylethyl Iodide); Coumarin 6(3-(2′-Benzothiazolyl)-7-dirnethylaminocournarin); Styryl 9M(2-(6-(4-Dimethylaminophenyl)-2,4-neopentrlene-1,3,5-hexatrienyl)-3-methyi-benzothiazoiium Perchlorate); Styryl15(2-(6-(9-(2,6,7-Tetrahydro-1H,5H-benzo(ij)-chinolizmium))-2,4-neopentyiene-1,3,5-hexatrienyl)-3-methyibenzothiazoliumPerchlorate); Styryl 14 (2-(8-(4-p-DimethyiammophenylVmethyibenzothiazoiium Perchlorate); Styryl 20(2-(8-(9-(2,3,6,7-Tetrahydro-1H,5H-benzo(ij)-chinolizinium))-2,4-neopentylene-1,3,5,7-octatraenyl)-3-methylbenzothiazoliumPerchlorate); Furan 1(Benzofuran,2,2′-[1,1′-biphenyl]-4,4′-diyl-bis-tetrasulfonic acid(tetrasodium salt)); and PBBO (2-(4-Biphenylyl)˜6-phenylbenzoxazoi˜1,3).

In some embodiments, the fluorescent moiety is a substituted stilbene.Examples of substituted stilbenes include but are not limited to DPS(4,4′-Diphenylstilbene); Stilbene 1 ([1,1′-Biphenyl]-4-sulfonic acid,4′,4″-1; 2-ethene-diylbis-, dipotassium salt); and Stilbene 3(2,2′-([1,1′-Biphenyl]-4,4′-diyldi-2,1ethenediyl)-bis-benzenesulfonicacid disodium salt). In some embodiments, the labeling agent contains afunctional group that rapidly reacts with amines, a fluorescent moiety,and optionally one or more of a tertiary amino group or other MS activeatom. In some embodiments, the functional group that rapidly reacts withamines is Fluorol 7GA (2-Butyl-6-(butylamino)-1H benz[de]isoquinoline-1,2(2H)-dione. Sulforhodamine B (Ethanaminium,N-[(6-diethylamino)-9-(2,4-disulfophenyl)-3H-xanthen-3-ylidene]-Nethylhydroxid, inner salt, sodium salt); and Sulforhodamine 101(8-(2,4-Disulfophenyl)-2,3,5,6,11,12,14,15-octahydro-1H,4H, 10H,13H-diquinolizino [9,9a, 1-bc:9′,9a′, 1-hi]xanthenes).

In some embodiments, the fluorescent moiety in the labeling agent is apyrromethene, e.g., Pyrromethene 546(1,3,5,7,8-pentamethylpyrromethenedifluoroborate complex); Pyrromethene556(Disodium-1,3,5,7,8-pentamethylpyrromethene-2,6-disulfonate-difluoroboratecomplex);

(Disodium-1,3,5,7,8-pentamethylpyrromethene-2,6-disulfonate-difluoroboratePyrromethene 567(2,6-Diethyl-1,3,5,7,8-pentamethylpyrromethenedifluoroborate complex);Pyrromethene 580(2,6-Di-n-butyl-1,3,5,7,8-pentamethylpyrromethenedifluoroboratecomplex); Pyrromethene 597 (2,6-Di-t-butyl-1,3,5,7,8-pentamethylpyrromethenedifluoroborate complex); and Pyrromethene 650(8-Cyano-1,2,3,5,6,7-hexamethylpyrromethenedifluoroborate complex).

In certain embodiments, the fluorescent moiety in the labeling agent isa pyrene-derivative, e.g., N-(1-pyrene)maleimide, or Pyranine (trisodium8-hydroxypyrene-1,3,6-trisulfonate).

In certain embodiments, the labeling reagent comprises a tertiary aminogroup or other MS active atom, and a functional group that rapidlyreacts with amines (such as an isocyanate, or succidimidylcarbamate). Incertain embodiments, the amino group or MS active group gives increasesdetectability of the glycan by MS. In particular embodiments, thefluorescent moiety provides a good fluorescence signal; and the reactivefunctional group gives rapid tagging of desired biomolecules. In certainembodiments, a fluorescent label and or a labeling reagent includes aquinolinyl fluorophore. In particular embodiments, the fluorescent labelcomprises a carbamate tagging group. In some embodiments, thefluorescent label and/or the labeling comprises a basic tertiary amine.In particular embodiments, the fluorophore comprises the carbamatetagging group, the quinolone fluorophore, and the tertiary amine.

In some embodiments, the glycans are contacted, treated, and/orincubated with a derivatizing or labeling reagent, for, for about, orfor at least 1 minute, 2 minutes, 3 minutes, 5 minutes, 10 minutes, 15minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45minutes, 50 minutes, 55 minutes, 60 minutes, 1.5 hours, 2 hours, 3hours, 4 hours, 5 hours, 6 hours, or between 1 minute and 1 hour,between 1 minute and 30 minutes, between 1 minute and 10 minutes,between 5 minutes and 15 minutes, or between 15 minutes and 30 minutes,between 1 hour and 6 hours, between 3 hours and 12 hours, or between 12hours and 48 hours, inclusive. In some embodiments, the glycan arecontacted and/or treated with the labeling and/or derivatizing reagentin the presence of a solvent, e.g., an organic solvent. In someembodiments, the solvent is hydrophilic. In certain embodiments, thesolvent is an aprotic solvent. In particular embodiments, the solvent isan amide, e.g., an organic amide, a sulfonamide, a phosphoramide, or aformamide. In some embodiments, the labeling reagent and/or derivatizingreagent is contacted, treated, and/or incubated with the glycans in thepresence of a solvent that is a formamide. In particular embodiments,the labeling reagent and/or derivatizing reagent is contacted, treated,and/or incubated with the glycans in the presence of adimethylformamide.

In some embodiments, the glycans are contacted, treated, and/orincubated with the derivatizing or labeling reagent at a temperature. Insome embodiments, the temperature is between 10° C. and 45° C., orbetween 24° C. and 37° C., or between 23° C. and 27° C. In someembodiments, the temperature is at least or at least about or is or isabout 10° C., 15° C., 20° C., 24° C., 25° C., 26° C., 27° C., 28° C.,29° C., 30° C., 31° C., 32° C., or any range between any of theforegoing. In certain embodiments, the glycans are contacted, treated,or incubated with the derivatizing or labeling reagent at roomtemperature, e.g., between 23° C. and 27° C., between 24° C. and 26° C.,or at or about 24° C., 25° C., or 26° C.

In some embodiments, the glycans are incubated, treated, and orcontacted with a derivatizing or labeling reagent. In some embodiments,the derivatizing or labeling reagent is 2-aminobenzamide (2-AB),2-aminobenzoic acid (2-AA), 2-aminopyridine (PA), 2-Aminoacridone(AMAC), 2-aminonaphthalene trisulfonic acid (ANTS), and1-aminopyrene-3,6,8-trisulfonic acid (APTS),3-(Acetylamino)-6-aminoacridin (AA-Ac), 6-Aminoquinoline (6-AQ),7-Aminomethyl-coumarin (AMC), 2-Amino (6-amido-biotinyl) pyridine (BAP),9-Fluorenylmethoxycarbonyl (FMOC)-hydrazide,1,2-Diamino-4,5-methylenedioxy-benzene (DMB), or o-Phenylenediamine(OPD). In some embodiments, the glycans are incubated with thederivatizing or labeling reagent for or for about 5 minutes at roomtemperature, e.g., between 23° C. and 27° C., between 24° C. and 26° C.,or at or about 24° C., 25° C., or 26° C.

In certain embodiments, the derivation and/or labeling reaction thatoccurs when the glycans are contacted, incubated, and/or treated withthe derivation and/or labeling reagent is ended by quenching thereaction. In some embodiments, the reaction is quenched by adding aquenching agent. In particular embodiments, the derivation and/orlabeling reaction is quenched prior to any analysis or quantification ofthe glycans, e.g., by chromatography and/or mass spectrometrytechniques. Suitable agents to quench derivatization and labelingreactions are known, and in some aspects will depend on the specificderivatization or labeling reaction. In some aspects, the derivationand/or labeling reagent is 2-aminobenzamide (2-AB), 2-aminobenzoic acid(2-AA), 2-aminopyridine (PA), 2-Aminoacridone (AMAC), 2-aminonaphthalenetrisulfonic acid (ANTS), and 1-aminopyrene-3,6,8-trisulfonic acid(APTS), 3-(Acetylamino)-6-aminoacridin (AA-Ac), 6-Aminoquinoline (6-AQ),7-Aminomethyl-coumarin (AMC), 2-Amino (6-amido-biotinyl) pyridine (BAP),9-Fluorenylmethoxycarbonyl (FMOC)-hydrazide,1,2-Diamino-4,5-methylenedioxy-benzene (DMB), or o-Phenylenediamine(OPD), and the reaction is quenched by adding a nitrile, e.g., anorganic nitrile. In some embodiments, the reaction is quenched withacetonitrile.

2. Purifying Glycans

In particular embodiments, a sample of the extracellular solution thatcontains the glycans is prepared for analysis, e.g., mass spectrometryanalysis. In some embodiments, the sample of glycans, e.g. N-glycans, ispurified prior to the analysis. In some embodiments, the purificationincludes any method capable of separating N-glycans from any entitieswhich will or will potentially disrupt, hinder, and/or weaken thedetection of the N-glycans. In some embodiments, the purification stepis performed to remove the N-glycans from cellular debris,deglycosylated protein, PNGase F, buffer/formulation components,surfactants, labeling reaction byproducts, and/or excess labeling and/orderivatization reagents. In particular embodiments, the purificationstep is performed on labeled glycans, e.g. N-glycans, e.g., glycans withcovalently attached detectable labels. In certain embodiments, thepurification is performed by any suitable technique for purifyingglycans, including but not limited to solid phase extraction (SPE),liquid-liquid extraction, gel filtration, paper chromatography, andprecipitation.

In some embodiments, the purification is or includes a solid phaseextraction (SPE). In particular embodiments, SPE is a sample preparationprocess by which compounds that are dissolved or suspended in a liquidmixture are separated from other compounds in the mixture, for example,according to their physical and/or chemical properties. In certainembodiments, chemical derivatization, e.g., addition of a detectablelabel, is performed prior to the analysis of the N-glycans, and SPE isperformed to remove reagents used for the derivation from the N-glycansprior to detection. In certain embodiments, N-glycans that are dissolvedor suspended in a liquid mixture are separated from other compounds inthe mixture. In particular embodiments, the SPE is a process thatutilizes the affinity of solutes, e.g., glycans, dissolved or suspendedin a liquid (i.e., a mobile phase) for a solid through which the sampleis passed (i.e., a stationary phase) to separate a mixture into desiredand undesired components. In some embodiments, the desired components,i.e., glycans, are retained on the stationary phase. In particularembodiments, undesired components of the sample are retained on thestationary phase. In certain embodiments, the glycans, e.g., N-glycans,are retained on the stationary phase and the solution that passesthrough the stationary phase is discarded. In particular embodiments,the undesired components are retained on the stationary phase and thesolution that passes through the stationary phase contains the glycans,e.g., N-glycans, and is collected. In some embodiments, the stationaryphase retains the glycans, which are then removed from the stationaryphase by contacting, passing, and/or rinsing the solid phase with aneluent.

In some embodiments, glycans, e.g. N-glycans, that have undergonederivation are purified prior to detection. In some embodiments, thepurification is performed by solid phase extraction (SPE). In someembodiments, SPE is performed on glycans after the derivation process.In certain embodiments, salts, e.g., excess salts from the derivationreaction or reactions, are removed from the glycans, e.g. N-glycans, bySPE. In certain embodiments, the glycans, e.g. N-glycans, undergo SPEafter a derivation reaction. In some embodiments, SPE is performed aftera detectable label is added to the glycans, e.g. N-glycans, e.g., by wayof a reductive amination reaction or hydrazide labeling. In someembodiments, SPE removes excess labeling reagent from the derivationreaction or reactions from the glycan sample.

In some embodiments, the solid phase of the SPE is or includes acartridge. In certain embodiments, the solid phase retains, collects,and/or binds glycans contains a polyamide adsorbent that may be used toadsorb glycans from aqueous solutions. In some embodiments, solutionscan be passed through these cartridges by using either gravity, vacuum,or positive pressure. In particular embodiments, the cartridge extractsglycans from other materials, e.g., salts, moieties, reagents, and/orcellular debris, according to a difference in hydrophobicity of theN-glycans and the other materials. In some embodiments, the SPEmaterials for preparation are C18 and charcoal, both of which have highselectivity for glycans.

In some embodiments, the SPE is performed with a commercially availablekit. In certain embodiments, the SPE is performed according to themanufacturer's instructions. Suitable kits that are of include SPE ofN-glycans include, but are not limited to GLYCOCLEAN H Cartridges(GKI-4025 ProZyme), ADVANCE BIO N-Glycan Deglycosylation cleanupcartridges (Agilent), Ludger Clean T1 cartridges (LC-T1-A6, Ludger),GlycoWorks 2-AB N-Glycan Kits (Waters), and GlycoWorks RapiFluor-MSN-Glycan Kit (Waters).

In some embodiments, the SPE is performed by a hydrophilic interactionliquid chromatography (HILIC) SPE process. In some embodiments, the SPEis performed by first conditioning the sorbent with water and thenequilibrating it to high acetonitrile loading conditions. In certainembodiments, the N-glycan samples are diluted with acetonitrile and areloaded and washed free of the sample matrix using an acidic washsolvent. In some embodiments, the columns, tubes, cartridges, or wellsfor use with the SPE process are conditioned with water, e.g., distilledwater. In some embodiments, the columns, tubes, cartridges, or wells areconditioned with an organic solvent, e.g., acetonitrile. In someembodiments, a sample or solution containing the glycans is loaded ontothe column, tube, cartridge, or well. In particular embodiments, thecolumns, tubes, cartridges, or wells containing the glycans are washedand/or rinsed at least once, twice, three times, four times, five times,six times, seven times, eight times, nine times, or ten times. Inparticular embodiments, the columns, tubes, cartridges, or wellscontaining the glycans are washed and/or rinsed with a solution. In someembodiments, the solution is or includes water. In particularembodiments, the solution is or includes acetonitrile. In particularembodiments, the solution is or includes formic acid. In certainembodiments, the columns, tubes, cartridges, or wells are rinsed and/orwashed in a solution that includes water, acetonitrile, and formic acid.

In some embodiments, the glycans are eluted from the columns, tubes,cartridges, or wells. In certain embodiments, the glycans are elutedwith a solution that is or includes ammonium acetate. In particularembodiments, the glycans are eluted with a solution that is or includesacetonitrile. In particular embodiments, the glycans are eluted with asolution that includes ammonium acetate and acetonitrile. In particularembodiments, the glycans are eluted with a solution that contains,contains about, or contains at least 1 mM, 5 mM, 10 mM, 20 mM, 50 mM,100 mM, 150 mM, 200 mM, 250 mM, 300 mM, 400 mM, 500 mM, or 1,000 mMammonium acetate. In certain embodiments, the glycans are eluted with asolution that contains, contains about, or contains at least 0.1%, 0.5%,1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, or 50%acetonitrile. In particular embodiments, the glycans are eluted with asolution that contains or contains about 200 mM ammonium acetate and 5%acetonitrile.

In some embodiments, the washing condition achieves optimal SPEselectivity by introducing electrostatic repulsion between theaminopropyl HILIC sorbent and reaction byproducts and by enhancing thesolubility of the matrix components. In particular embodiments, thelabeled, released glycans are next eluted from the HILIC sorbent. Incertain embodiments, the SPE sorbent has a weakly basic surface, and thecapacity for anion exchange, just as it has the capacity for cationrepulsion, and so the labeled glycans are eluted with an eluent ofsignificant ionic strength. Thus, in some embodiments, the elutionbuffer comprises a pH 7 solution of 200 mM ammonium acetate in 5%acetonitrile. In certain embodiments, upon their elution, the labeledglycans can be diluted with a mixture of organic solvents e.g.,acetonitrile and dimethylformamide, and directly analyzed by UPLC orHPLC HILIC column chromatography using fluorescence and/or ESI-MSdetection.

In some embodiments, the amount or concentration of glycans, e.g.N-glycans in a sample, solution, and/or media is adjusted. In particularembodiments, the amount or concentration of glycans, e.g. N-glycans in asample, solution, and/or media is adjusted by evaporating the sample,solution, and/or media, and resuspending the glycans, e.g. N-glycans, ina solution to the desired concentration. In some embodiments, the volumeof a sample, solution, and/or media that contains the glycans, e.g.N-glycans, is adjusted by evaporating the sample, solution, and/ormedia, and resuspending the glycans in a solution of the desired volume.In some embodiments, the sample, solution, and/or media is evaporated byvacuum centrifugation.

In particular embodiments, the sample, solution, and/or media containingglycans, e.g. N-glycans, is evaporated prior to a purification step,e.g., SPE. In some embodiments, the sample, solution, and/or mediacontaining glycans, e.g. N-glycans, is evaporated by vacuumcentrifugation prior to a purification step. In some embodiments, thesample, solution, and/or media containing glycans, e.g. N-glycans, isevaporated following the purification step. In particular embodiments,the sample, solution, and/or media containing glycans, e.g. N-glycans,is evaporated by vacuum centrifugation following a purification step. Incertain embodiments, the sample, solution, and/or media containingglycans, e.g. N-glycans, is evaporated prior to detection, e.g.,detection by HPLC and/or MS. In some embodiments, the sample, solution,and/or media containing glycan, e.g. N-glycans, is evaporated by vacuumcentrifugation prior to detection. In some embodiments, the vacuumcentrifugation allows for the sample containing glycans to alter oradjust volumes for different steps or procedures. For example, volumemight be increased prior to purifying the N-glycans with SPE so thatunwanted materials, e.g., cellular debris, does not clog the SPE columnor cartridge. In such instances, the eluate containing the glycans maybe vacuum centrifuged and resuspended in a smaller volume, for examplefor analysis with HPLC or mass spectrometry.

3. Profiling the Glycans

Provided are methods for assessing cell surface glycans, e.g.,N-glycans, by determining the presence, absence, or level of the glycanspresent in a sample. In particular embodiments the sample containsglycans that have been released, removed, or detached from the surfaceof cells present in a cell composition, e.g., a test cell composition.In certain embodiments, a sample containing glycans, e.g., N-glycans,that have been released from the surface of cells are assessed and oranalyzed to determine the presence, absence, or level of glycans presentin the sample.

In some embodiments, isolated and/or released surface glycans may beanalyzed or assessed by any known technique in the art that is suitablefor the detection, analysis, and or isolation of glycans, e.g.,N-glycans. For example, in certain embodiments, glycans are analyzedusing one or more available methods described in Anumula, Anal. Biochem.350(1):1, 2006; Klein et al., Anal. Biochem., 179:162, 1989; and/orTownsend, R. R. Carbohydrate Analysis High Performance LiquidChromatography and Capillary Electrophoresis., Ed. Z. El Rassi, pp181-209, 1995. For example, in some embodiments, glycans arecharacterized using one or more of chromatographic methods,electrophoretic methods, nuclear magnetic resonance methods, andcombinations thereof. Exemplary such methods include, for example, NMR,mass spectrometry, liquid chromatography, 2-dimensional chromatography,SDS-PAGE, antibody staining, lectin staining, monosaccharidequantitation, capillary electrophoresis, fluorophore-assistedcarbohydrate electrophoresis (FACE), micellar electrokineticchromatography (MEKC), exoglycosidase or endoglycosidase treatments, andcombinations thereof. Those of ordinary skill in the art will be awareof other methods that can be used to characterize glycans. In certainembodiments, isolated surface glycans are analyzed or assessed by atechnique that is or includes liquid chromatography, fluorescencedetection, and/or mass spectrometry.

a. Chromatography

In some embodiments, liquid chromatography (LC), including highperformance liquid chromatography, is used to analyze glycans (e.g.,N-glycans), that are present in the sample. Various forms of LC can beused to study glycans, including anion-exchange chromatography,reversed-phase HPLC, size-exclusion chromatography, high-performanceanion-exchange chromatography, and normal phase (NP) chromatography,including NP-HPLC. Hydrophilic interaction chromatography (HILIC) is avariant of NP-HPLC that can be performed with partially aqueous mobilephases, permitting normal-phase separation of peptides, carbohydrates,nucleic acids, and many proteins. In some embodiments, glycans in asample are detected by, or by methods that include, LC, e.g., HPLC orHILIC.

In certain embodiments, the liquid chromatography is a high performanceliquid chromatography (HPLC), ultra-high performance liquidchromatography (UHPLC), or ultra-performance liquid chromatography(UPLC). In some embodiments, HPLC is distinguished from traditional(“low pressure”) liquid chromatography because operational pressures aresignificantly higher, 50-350 bar (725-5070 psi), while ordinary liquidchromatography typically relies on the force of gravity to pass themobile phase through the column. UHPLC operate at pressures of up to1030 bar (15,000 psi).

In some embodiments, glycans in a sample are detected by, or by methodsthat include, HILIC. In some embodiments, HILIC is a form of HPLC thatcan be used in the methods described herein. HILIC separates analytesbased on polar interactions between the analytes and the stationaryphase (e.g., substrate). The polar analyte associates with and isretained by the polar stationary phase. Adsorption strengths increasewith increase in analyte polarity, and the interaction between the polaranalyte and the polar stationary phase (relative to the mobile phase)increases the elution time. Use of more polar solvents in the mobilephase will decrease the retention time of the analytes while morehydrophobic solvents tend to increase retention times. The elution orderfor HILIC is least polar to most polar, the opposite of that inreversed-phase HPLC. In some embodiments, HILIC can be performed on anHPLC system.

In particular embodiments, glycans are detected by or by methods thatinclude HILIC. In some embodiments, various types of substrates can beused with HILIC, e.g., for column chromatography, including silica,amino, amide, cellulose, cyclodextrin and polystyrene substrates.Examples of useful substrates, e.g., that can be used in columnchromatography, include but are not limited to: polySulfoethylAspartamide (e.g., from PolyLC), a sulfobetaine substrate, e.g.,ZIC®-HILIC (e.g., from SeQuant), POROS® HS (e.g., from AppliedBiosystems), POROS® S (e.g., from Applied Biosystems), PolyHydroethylAspartamide (e.g., from PolyLC), Zorbax 300 SCX (e.g., from Agilent),PolyGLYCOPLEX® (e.g., from PolyLC), Amide-80 (e.g., from Tosohaas), TSKGEL® Amide-80 (e.g., from Tosohaas), Polyhydroxyethyl A (e.g., fromPolyLC), Glyco-Sep-N (e.g., from Oxford GlycoSciences), and AtlantisHILIC (e.g., from Waters). Preferred columns include polySulfoethylAspartamide and ZIC®-HILIC; the most preferred column beingpolySulfoethyl Aspartamide. Column that can be used in the disclosedmethods include columns that utilize one or more of the followingfunctional groups: carbamoyl groups, sulfopropyl groups, sulfoethylgroups (e.g., poly (2-sulfoethyl aspartamide)), hydroxyethyl groups(e.g., poly (2-hydroxyethyl aspartamide)) and aromatic sulfonic acidgroups. Preferred functional groups include sulfoethyl groups such aspoly (2-sulfoethyl aspartamide) and sulfopropyl groups such asCH₂N(CH₃)₂CH₂CH₂CH₂SO₃.

In some embodiments, the LC analyzed glycans are then further subjectedto analysis by mass spectrometry. Examples of mass spectrometry that canbe used to further analyze the glycans include ESI-MS, turbosprayionization mass spectrometry, nanospray ionization mass spectrometry,thermospray ionization mass spectrometry, sonic spray ionization massspectrometry, SELDI-MS and MALDI-MS. For example, the methods describedherein can be used to provide LC-evaluated glycans for on-line massspectrometry (e.g., ESI-MS) and/or for off-line mass spectrometry (e.g.,MALDI-MS) without further purification.

In certain embodiments, glycans are labeled with a chromophore or afluorophore allow for sensitive detection in HILIC. In some embodiments,the most commonly used labels are PA, 2-AB, and 2-AA, but other tagssuch as 3-(acetylamino)-6-aminoacridine, or other tags such as tagscontaining a the quinolone fluorophore, and the tertiary amine are alsouseful. In particular embodiments, glycans that are labeled with tagscontaining a quinolone fluorophore and the tertiary amine are detectedby or by methods that include HILIC. As most of these labels havehydrophobic characteristics, the derivatized glycans may show slightlyless retention than native, unlabeled glycans. In some embodiments,fluorescence detection is performed using detectors equipped with axenon lamp and both excitation and emission monochromators can be set tothe optimal wavelengths of the chosen label to achieve high sensitivity.

b. Fluorescence Detection

In certain embodiments, fluorescence detection is coupled to a liquidchromatography and/or a mass spectrometry technique. In certainembodiments, the glylcan, e.g., an N-glycan, is attached, e.g.,covalently attached, to a fluorescent label. In particular embodiments,the fluorescently labeled glycan, e.g., an N-glycan, is analyzed byfluorescence detection with liquid chromatography followed by analysiswith mass spectrometry. In some embodiments, the combination of liquidchromatography, mass spectrometry, and fluorescent detection is ananalytical platform that can be used for a comprehensive glycan analysisof a sample, e.g., a sample of N-glycans that were released or detachedfrom the surface of cells.

In some embodiments, HPLC-fluorescence detection has a number ofbenefits, including high sensitivity, high selectivity, andrepeatability. The most advanced fluorescence detectors feature atemperature-controlled cell to ensure stable analysis even if theambient temperature fluctuates. These detectors also provide high levelsof sensitivity and validation to support functions in a wide range ofapplications from conventional to ultra-fast LC analysis. In certainembodiments, the glycans are analyzed with a fluorescence detector thatfeatures a temperature-controlled cell.

In some embodiments, fluorescence labeling is used with liquidchromatography and/or mass spectrometry to detect glycans. Many organicmolecules, e.g., glycans, exhibit strong UV absorbance at wavelengthsless than 210 nm, thus allowing a detector set at 200 nm to act somewhatas a “universal” detector. Fluorescence is much less common than UVabsorbance. Thus, in some embodiments, given a sample containing anamount of unwanted material or impurities, fluorescence detection wouldonly measure tagged or labeled molecules, thus overcoming issues ofbackground or selectivity. In some embodiments, fluorescence detectorscan enhance the selectivity of the glycan analysis.

In some embodiments, a sample of glycans, e.g., N-glycans released fromthe surface of cells, is labeled with a fluorescent label, and morespecies of glycans are detected than are detected than with a sample ofglycans are not labeled. In some embodiments, a sample of glycans islabeled with a fluorescent label, and at least 10%, at least 20%, atleast 30% at least 40%, at least 50%, at least 60%, at least 70%, atleast 80%, at least 90%, at least 95%, at least 100%, or is at least1-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least5-fold, or at least 10-fold more species of glycans in the sample aredetected than with a sample of glycans that are not labeled.

In certain embodiments, a sample of glycans, e.g., N-glycans releasedfrom the surface of cells, is labeled with a fluorescent label, and theglycans are detected with a greater sensitivity than the glycans are notlabeled. In certain embodiments, the sensitivity for detection isincreased by at least 10%, at least 20%, at least 30% at least 40%, atleast 50%, at least 60%, at least 70%, at least 80%, at least 90%, atleast 95%, at least 100%, or is at least 1-fold, at least 2-fold, atleast 3-fold, at least 4-fold, at least 5-fold, at least 10-fold, or atleast 100-fold when the N-glycans are labeled as compared to when theN-glycans are not labeled.

In some embodiments, glycans in a sample are selectively labeled with afluorescent label, and the sample is assessed with liquid chromatographythat is coupled with fluorescence detection. In some embodiments, theglycans are selectively labeled so that the only glycans contain theattached label in the sample. In certain embodiments, the glycans areselectively labeled so that glycans are labeled at least 2 times, 5times, 10 times, 50 times, 100 times, 1,000 times, 2,000 times, 5,000times, 10,000 times, 50,000 times, 100,000 times, 250,000 times, 500,000times, 1,000,000 or greater that the portion of sample that are notglycans, e.g., impurities such as proteins.

c. Mass Spectrometry

In various embodiments, methods comprise subjecting a portion of aglycan mixture to analysis with a mass spectrometric technique. Massspectrometry (MS), in its most simple definition, is the production anddetection of ions separated according to their mass-to-charge (m/z)ratios. The detection of such ions results in a mass spectrum which is aplot of the relative abundance of the ions as a function of their m/zratio. The two most widely-used MS ionization techniques for theanalysis of glycans are Matrix-Assisted Laser Desorption/Ionization(MALDI) and Electrospray Ionization (ESI); in both free glycans aretypically detected as metal (usually sodium) adducts in the positive ionmode, and as deprotonated or anion-adducted species in the negative-ionmode. Glycans may be detected in their protonated or deprotonated forms.Both MALDI and ESI are soft ionization techniques; i.e., the ionizationprocess imparts little excess energy and thus generates few or nofragments for glycans so that the intact molecular ions can be easilyobserved. Nevertheless, prompt loss of labile groups, especially sialicacid and fucose, may occur and the extent of its occurrence should beassessed, particularly in quantitative studies. Both MALDI-MS and ESI-MScan be applied to obtain an overall glycan profile.

Methods of the present inventions can be performed with a wide varietyof mass spectrometry instruments and techniques, including but notlimited to, matrix assisted laser desorption/ionization massspectrometry time of flight mass spectrometry (MALDI-TOF-MS),MALDI-TOF-TOF-MS, liquid chromatography with mass spectrometry (LC-MS),liquid chromatography with tandem mass spectrometry (LC-MS/MS), or bydirect infusion electrospray ionization mass spectrometry (ESI-MS). Insome embodiments, the N-glycans are detected by, or by a method thatincludes, analysis with MALDI-MS or ESI-MS.

In particular embodiments, N-glycans are detected with a high resolutionmass spectrometer. In some embodiments, the resolution of the massspectrometer is greater than 50 amu, 25 amu, 10 amu, 5 amu, 1 amu, 0.5amu, or 0.1 amu. In certain embodiments, the resolution of the massspectrometer is greater than 1 amu.

In some embodiments, mass spectrometry is performed without priorseparation by liquid chromatography. In particular embodiments, theglycans are detected without prior separation by liquid chromatographyby surface enhanced laser desorption ionization mass spectrometry(SELDI-MS) or matrix assisted laser desorption/ionization massspectrometry (MALDI-MS). In some embodiments, the glycans are detectedwithout prior separation by liquid chromatography by MALDI-MS.

MALDI is an ionization technique that uses a laser energy absorbingmatrix to create ions from large molecules with minimal fragmentation.It has been applied to the analysis of biomolecules (biopolymers such asDNA, proteins, peptides and sugars) and large organic molecules (such aspolymers, dendrimers and other macromolecules), which tend to be fragileand fragment when ionized by more conventional ionization methods. It issimilar in character to electrospray ionization (ESI) in that bothtechniques are relatively soft (low fragmentation) ways of obtainingions of large molecules in the gas phase, though MALDI typicallyproduces far fewer multiply charged ions. In certain embodiments,glycans in a sample, e.g., a sample of surface expressed glycans, aredetected by MALDI-MS. In some embodiments, the glycans are N-glycans.

In certain embodiments, liquid chromatography-mass spectrometry (LC-MS)is an analytical chemistry technique that combines the physicalseparation capabilities of liquid chromatography (or HPLC) with the massanalysis capabilities of mass spectrometry (MS). In some embodiments,coupled chromatography-MS (LC-MS) systems are employed because theindividual capabilities of each technique are enhanced synergistically.While liquid chromatography separates mixtures with multiple components,mass spectrometry provides structural identity of the individualcomponents with high molecular specificity and detection sensitivity. Insome embodiments, glycans are detected by liquid chromatography, e.g.,HILIC HPLC, followed by mass spectrometry. In some embodiments, the massspectrometry is electrospray ionization mass spectrometry (ESI-MS),turbospray ionization mass spectrometry, nanospray ionization massspectrometry, thermospray ionization mass spectrometry, sonic sprayionization mass spectrometry.

In certain embodiments, the glycans, e.g., N-glycans, are detected byESI-MS after sorting by liquid chromatography. In such embodiments,analysis by liquid chromatography, e.g., with fluorescence detection,provides information of the relative abundance of an N-glycan species,e.g., by calculating the area under the curve corresponding to theindividual glycan, and expressing it as a ratio or a percentage of thetotal areas of all the curve corresponding to glycans. In certainembodiments, standards can be used to determine to amount of the glycanpresent in the sample. In certain embodiments, the LC, e.g., HILIC, ison-line with the mass spectrometer. In some embodiments, the massspectrometer measures the precise mass of the N-glycans, allowing forthe identification of the glycans that correspond to the peaks detectedby the LC. In some embodiments, glycans, e.g., N-glycans, are assessedfor the presence, absence, or amount by LC-MS.

In particular embodiments, the glycans, e.g., N-glycans, that have beenremoved from a cellular surface are detected with by LC-ESI-MS. ESI is atechnique used in mass spectrometry to produce ions using anelectrospray in which a high voltage is applied to a liquid to create anaerosol. In some embodiments, ESI is useful in producing ions frommacromolecules by overcoming the propensity of these molecules tofragment when ionized. ESI is different from other atmospheric pressureionization processes (e.g. MALDI) since it may produce multiple chargedions, effectively extending the mass range of the analyzer toaccommodate the kDa-MDa orders of magnitude observed in proteins andtheir associated polypeptide fragments.

In certain embodiments, the mass spectrometry is tandem massspectrometry (tandem MS or MS/MS). In particular embodiments,MALDI-TOF-MS, MALDI-TOF-TOF-MS, LC-MS, and/or ESI-MS techniques areperformed with tandem MS and/or are performed with a tandem MStechnique. In particular embodiments, tandem MS involves more thanone-step of mass selection or analysis, and fragmentation is usuallyinduced between the steps. In some embodiments, tandem mass spectrometryis used and/or employed for glycan identification, quantification,and/or detection. The first mass analyzer, i.e., first stage MS,isolates ions of a particular m/z value that represent a single speciesof glycan among many introduced into and then emerging from the ionsource. Those ions, e.g., ionized glycan particles, are then fragmented,e.g., accelerated into a collision cell containing an inert gas such asargon to induce ion fragmentation. This process is designated“collisionally induced dissociation” (CID) or “collisionally activateddissociation” (CAD). The m/z values of fragment ions, e.g., ionizedglycan fragments, are then measured in a 2^(nd) mass analyzer, i.e.,second stage MS, to obtain structural information. In particularembodiments, glycans are identified, quantified, and/or detected bytandem MS.

There are several types of tandem mass spectrometers, including triplestage quadrupoles (TSQ), 3D and linear ion traps,quadrupole/time-of-flight (QTOF) hybrid instruments, quadrupole-linearion trap hybrid instruments (QTRAP), and time-of-flight-time-of-flight(TOF/TOF) instruments. With 3D or linear quadrupole ion traps, tandem MScan also be performed in a single mass analyzer over time and in theseinstruments this process may be iterated more than once to yield MS^(n)spectra. These instruments achieve fragmentation by resonanceexcitation, which induces collisions with the trap bath gas (helium) ofsufficiently high energy to induce fragmentation. The other instrumentsmentioned above employ CID in a collision cell. Other methods that canbe used to fragment molecules for tandem MS include electron capturedissociation (ECD), electron transfer dissociation (ETD), infraredmultiphoton dissociation (IRMPD) and blackbody infrared radiativedissociation (BIRD) (9).

In some embodiments, particles are fragmented between the first stageand the second stage MS. In particular embodiments, the fragmentation isin-source fragmentation, collision-induced fragmentation,collision-induced dissociation, election capture dissociation, electrontransfer dissociation, negative electron transfer dissociation,electron-detachment dissociation, charge transfer dissociation,photodissociation, infrared multiphoton dissociation, blackbody infraredradiative dissociation, surface induced dissociation.

In particular embodiments, the MS is tandem MALDI-MS (MALDI-MS/MS) ortandem ESI-MS (ESI-MS/MS). In certain embodiments, the N-glycans arequantified, detected, and/or identified by, or by a method thatincludes, analysis with tandem MALDI-MS or tandem ESI-MS.

4. Profiles and Maps

Provided herein are methods for assessing and/or analyzing a samplecontaining glycans, e.g., a sample containing released surface N-glycansfrom a cell composition, e.g. test cell composition. In particularembodiments, the sample is assessed or analyzed by determining thepresence, absence, level, relative abundance, and/or amount of one ormore glycans, e.g. N-glycan. In some embodiments, the one or moreglycans, e.g. N-glycans, are part of a cell surface profile of glycansdetected in the sample. In some embodiments, the cell surface profileindicates the presence, absence, level, relative abundance and/or amountof one or more different species of N-glycans. In some aspects, the oneor more different species can include any as set forth in Table E1 or asubset thereof. In some embodiments, the number of species of differentglycans is greater than or greater than about 5, 10, 20, 30, 40, 50, 60,70, 80, 90, 100, 150, 200, 300, 400, 500 or more different species.

In some aspects, the one or more different species of N-glycans include,for example, high mannose N-glycans, bisected and Sialyl Lewis^(X)N-glycans, and/or N-acetyl lactosamine containing N-glycans.

In some aspects, the one or more different species of N-glycans include,for example, a fucosylated biantennary complex glycan having no reducingend terminal galactose residues, a fucosylated biantennary complexglycan having one reducing end terminal galactose residue, a fucosylatedbiantennary complex glycan having two reducing end terminal galactoseresidues, a biantennary complex glycan having no reducing end terminalgalactose residues, a biantennary complex glycan having one reducing endterminal galactose residue, a biantennary complex glycan having tworeducing end terminal galactose residues, a fucosylated biantennarycomplex glycan having two galactose residues and one N-acetylneuraminicacid residue, a fucosylated biantennary complex glycan having twogalactose residues and two N-acetylneuraminic acid residues, abiantennary complex glycan having two galactose residues and twoN-acetylneuraminic acid residues, a high mannose glycan having fivemannose residues, a high mannose glycan having six mannose residues, ahigh mannose glycan having seven mannose residues, a high mannose glycanhaving eight mannose residues, and/or a high mannose glycan having ninemannose residues.

In some embodiments, the sample is assessed for the presence, absence,level, relative abundance and/or amount of a target glycan or one ormore target glycans. In certain embodiments, least 10, at least 20, atleast 25, at least 30, at least 40, at least 50, at least 60, at least70, at least 80, at least 90, at least 100, at least 125, at least 150,at least 175, at least 200, at least 225, at least 250, at least 300, atleast 400, or more than 500 different glycan species are assessed oranalyzed, e.g., for the presence, absence, relative level, or amount ofthe glycans.

In some embodiments, the target glycan or glycans are those whosepresence, absence, level, relative abundance and/or amount on the cellsurface is associated with one or more functional and/or phenotypicactivity. In some cases, the target glycan or glycans is one that isassociated with or that may affect or impact or alter an activity formamong masking of a cell surface marker, a metabolic activity,differentiation state, proliferative or expansion capacity, activationstate, cytolytic activity, signaling activity, an adhesion property, ora homing property.

In some aspects, the one or more different species can include any asset forth in Table E1 or a subset thereof. In some embodiments, thenumber of species of different glycans is greater than or greater thanabout 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400,500 or more different species.

In some aspects, the one or more different species of N-glycans include,for example, high mannose N-glycans, bisected and Sialyl Lewis'N-glycans, and/or N-acetyl lactosamine containing N-glycans.

In some aspects, the one or more different species of N-glycans include,for example, a fucosylated biantennary complex glycan having no reducingend terminal galactose residues, a fucosylated biantennary complexglycan having one reducing end terminal galactose residue, a fucosylatedbiantennary complex glycan having two reducing end terminal galactoseresidues, a biantennary complex glycan having no reducing end terminalgalactose residues, a biantennary complex glycan having one reducing endterminal galactose residue, a biantennary complex glycan having tworeducing end terminal galactose residues, a fucosylated biantennarycomplex glycan having two galactose residues and one N-acetylneuraminicacid residue, a fucosylated biantennary complex glycan having twogalactose residues and two N-acetylneuraminic acid residues, abiantennary complex glycan having two galactose residues and twoN-acetylneuraminic acid residues, a high mannose glycan having fivemannose residues, a high mannose glycan having six mannose residues, ahigh mannose glycan having seven mannose residues, a high mannose glycanhaving eight mannose residues, and/or a high mannose glycan having ninemannose residues. In some embodiments, the methods provided herein allowfor detection of relative levels of individual glycan species within apopulation of glycans. For example, in some embodiments, the area undereach peak of a liquid chromatograph can be measured and expressed as apercentage of the total. Such an analysis provides a relative percentamount of each glycan species within a population of surface expressedglycans.

In certain embodiments, methods provided herein facilitate detection ofglycans that are present at very low levels in the sample, e.g., asample containing surface expressed N-glycans. In certain embodiments,it is possible to detect and/or quantify the levels of glycans that arepresent at levels less than about 10%, 5%, 4%, 3%, 2%, 1.5%, 1%, 0.75%,0.5%, 0.25%, 0.1%, 0.075%, 0.05%, 0.025%, 0.01%, 0.001%, 0.0001%, or0.00001% within a population of glycans. In some embodiments, it ispossible to detect and/or optionally quantify the levels of glycans thatmake up between 0.1% and 10%, between 0.0001% and 0.1%, between 0.1% and1%, between 1% and 5%, between 0.00001% and 1%, between 0.1% and 2%, orbetween 0.1% and 1% of the total glycans in the sample.

In some embodiments, methods described herein allow for detection ofparticular linkages that are present at low levels within a sample ofsurfaced expressed glycans, e.g., N-glycans. For example, in someembodiments, the present methods allow for detection of particularlinkages that are present at levels less than 10%, less than 5%, lessthan 4%, less than 3%, less than 2%, less than 1.5%, less than 1%, lessthan 0.75%, less than 0.5%, less than 0.25%, less than 0.1%, less than0.075%, less than 0.05%, less than 0.025%, less than 0.01%, less than0.001%, less than 0.0001%, or less than 0.00001% of population ofsurface expressed glycans.

In certain embodiments, the identity of individual glycans within aprofile may be determined. Particular embodiments contemplate that theidentities of individual glycan species may be readily identified, forexample from the results of an analysis, e.g., by HPLC, UPLC,exoglycosidase sequencing and mass spectrometry (MALDI-MS, ESI-MS,ESI-MS/MS, LC-MS, LC-ESI-MS/MS). In some embodiments, the identity ofone or more glycans may be determined through the use of a referencedatabase. Several databases are available to assist with determiningglycan identity and/or glycan structure based on information obtainedfrom techniques including but not limited to HPLC, UPLC, exoglycosidasesequencing and mass spectrometry (MALDI-MS, ESI-MS, ESI-MS/MS, LC-MS,LC-ESI-MS/MS) data. In some embodiments, suitable databases include, butare not limited to, GlycoBase (glycobase.nibrtie/glycobase/shownibrtaction); Glycosciences(Glycosciences.de); UniCarbKB(unicarbkb.org); UniCarbDB (unicarb-db.biomedicine.gu.se); SugarBindDB(sugarbind.expasy.org), and Expasy (expasy.org/glycomics).

In particular embodiments, the methods provided herein allow for theanalysis and/or assessment of surface glycans in a sample. In certainembodiments, the methods provided herein allow for the detection,identification, and or quantification of levels of cell surface glycansthat are present in an amount of between about 0.1 fmol to about 1 mmolin a sample. In particular embodiments, a glycan that is present in asample in an amount of at least 10 amol, at least 100 amol, at least 500amol, at least 1 fmol, at least 5 fmol, or at least 10 fmol of a glycanspecies that is present in a sample of glycans can be detected,identified, and/or quantified by the methods provided herein.

C. Detecting Substances

In certain embodiments, the methods provided herein are used to detect apresence, absence, identity, or level of one or more substances, e.g.,residual substances, that may be present in a cell composition and/or ina sample collected from a cell composition. In certain embodiments, thesubstance is any compound, component, material, or matter that is orincludes one or more glycans. In particular embodiments, the substanceis or is produced, generated, expressed, and/or synthesized by a livingorganism. In certain embodiments, the substance is or includes a nucleicacid, protein, and/or lipid moiety. In particular embodiments, thesubstance is or includes one or more proteins, e.g., glycoproteins.

In some embodiments, methods for detecting the presence, absence,identity, and/or level of one or more substances in a cell compositionis or includes generating a surface N-glycan profile of from the cellcomposition and/or from a sample collected from the cell composition. Incertain aspects, the presence of a substance, e.g., a residualsubstance, is indicated by the identification and/or detection of aglycan that exogenous to, is not native to, and/or is not produced bythe cells in the cell composition, e.g., within the surface glycanprofile.

In certain embodiments, the non-native and/or exogenous glycan is notnative to the cells of the composition, e.g., the glycan is notgenerated, synthesized, and/or produced by the cell and/or is notattached or bound to a protein expressed and/or synthesized by any ofthe cells of the composition. In certain embodiments, a glycan that isnative to a cell is a glycan that is synthesized by the cell. In someaspects, the glycan, e.g., N-glycan, biosynthesis occurs in the ER andthe Golgi, and, in some aspects, includes the assembly oligosaccharidesand transfer to amino acid residues, e.g., Asn, of proteins, such assecretory or membrane proteins, during translocation into the ER. Insome embodiments, the glycan biosynthesis is or includes furtherprocessing, such as by glycosidases and glycosyltransferases in thelumen of the ER, and, in some aspects, continues in the Golgi. Incertain embodiments, a glycan is non-native or exogenous to a cell ifthe glycan is not synthesized by and/or within the cell, e.g., at, near,or within the ER or Golgi.

In certain embodiments, the non-native and/or the exogenous glycan isnot produced, expressed, or otherwise present on the surface and/orbound to proteins at the surface, of cells from the same kingdom,phylum, class, order, family, genus, and/or species as the cells of thecomposition. Particular embodiments contemplate that for a givenspecies, e.g., human, an exogenous and/or non-native glycan may beidentified as a matter of routine. For example, in certain embodiments,the glycosylation and the identities of individual endogenous glycansvaries among different kingdom, phylum, class, order, family, genus,and/or species or organism. In some aspects, eukaryotic cells share theability to modify proteins by N-glycosylation. In some embodiments, thefirst steps of N-glycosylation in eukaryotic cells occurs in theendoplasmic reticulum (ER). In certain aspects, the glycosylationmachinery of the ER is highly conserved between all species and resultsin the biosynthesis of a common Man3GlcNAc2 core structure. Particularembodiments contemplate that further modifications of the N-glycan coretake place in the Golgi apparatus whereupon the glycosylation repertoirevaries among species. For example, in certain embodiments, yeast expresshigh-mannose glycan structures harboring up to 100 mannose residues indifferent linkages. In some embodiments, N-glycans found on insect cellproteins belong to the paucimannosidic type which represents the corestructure, with further modifications by additional mannose, fucose, andgalactose residues. Higher plants even synthesize a significant portionof complex type glycans with two antennae, and non-human immunogenicxylose residues occur with a high frequency. By contrast, animals mainlyexpress multiantennary complex type N-glycans and carry sialic acids atoutermost positions of glycan chains. However, in certain aspects,humans do not synthesize two of the major mammalian glycan epitopes,Gala1-3Gal (alpha-Gal) and N-glycolylneuraminic acid (Neu5Gc). In someaspects, humans commonly and/or typically have antibodies directedagainst these structures. In some embodiments, the non-native and/orexogenous glycan is not a mammalian glycan, e.g., a glycan that is notexpressed or synthesized by a mammalian cell. In certain embodiments,the glycan is not a human glycan, e.g., a glycan that is not produced bya human cell, such as a healthy human cell and/or a non-cancerous humancell and/or non-tumorigenic human cell.

In some embodiments, the exogenous and/or non-native glycan is from,produced, and/or synthesized by the same species as the cells of thecell composition. For example, particular embodiments contemplate thatwithin the same species, cells of different lineages, tissues, and/orstages of maturity may express or synthesize one or more differentglycans. Thus, in some embodiments, an exogenous and/or non-nativeglycan is produced by a cell of the same species but a different celltype as the cells in the cell composition. In some embodiments, thenon-native glycan is expressed and/or synthesized by cells of the samespecies but not by immune cells. In particular embodiments, thenon-native glycan is expressed and/or synthesized by human cells but notby human immune cells. In some embodiments, the non-native glycan isexpressed and/or synthesized by human cells but not by human T cells.

In some embodiments, the source of the non-native and/or exogenousglycan is a protein, e.g., a glycoprotein. In particular embodiments,the glycoprotein may include, but is not limited to, an antibody and/ora fragment or variant thereof, an MHC molecule and/or a fragment orvariant thereof, a growth factor, a cytokine, a chemokine, antibody, anFc-fusion protein, and/or an interleukin. In some embodiments, theglycoprotein is present in a solution, media, and/or a serum. In someembodiments, the glycoprotein is a recombinant protein. In someembodiments, the protein is not native, not endogenous, and/or is notexpressed by the cells of the cell composition.

In particular embodiments, the source of the non-native and/or exogenousprotein is one or more proteins, e.g., glycoproteins, that have beencontacted and/or exposed to the cells of the composition. In someembodiments, the one or more proteins have been contacted and/or exposedto the cells during a process for generating, producing, and/orengineering the cell composition. In some aspects, the one or more havebeen added, contacted, or incubated with the cells during the culturingof said cells, and/or during a process such as engineering, activating,stimulating, transducing, transfecting, cultivating, or expanding thecells, e.g., to produce or engineer the cell composition.

In some embodiments, the source of the exogenous and/or non-nativeglycan is a serum, e.g., one or more proteins found within the serum. Insome aspects, serum is commonly used as a supplement to cell culturemedia. In particular aspects, serum is used to provide one or more of abroad spectrum of macromolecules, carrier proteins for lipoid substancesand trace elements, attachment and spreading factors, low molecularweight nutrients, hormones and growth factors. In certain embodiments,the serum is animal serum. In certain embodiments, the source of thenon-native and/or exogenous glycan is or includes fetal bovine serum(FBS), bovine calf serum (BCS), newborn calf serum (NBCS), horse serum,goat serum, lamb serum, donkey serum, or porcine serum. In some aspects,the animal serum is fetal bovine serum (FBS). In some embodiments, thesource of the glycan is a serum and/or a protein, e.g., a glycoprotein,that is present in the serum. In some embodiments, the detection of anon-native and/or exogenous glycan indicates residual serum proteinswithin the cell composition.

In some embodiments, the source of the non-native and/or exogenousglycan is a recombinant protein. In some aspects, the recombinantprotein may be added to as a component of a serum replacement and/orserum alternative. In particular embodiments, the source of theexogenous and/or non-native glycan is a serum replacement and/or a serumalternative. In particular embodiments, serum replacement and/or serumalternative is defined, e.g., contains known identities and amount ofproteins, e.g., recombinant proteins. In some embodiments, the detectionof a non-native and/or exogenous glycan indicates residual recombinantproteins within the cell composition.

In some embodiments, the source of the exogenous and/or non-nativeglycan is a microorganism or virus. In certain aspects, virus, yeast,mold, mycoplasma, contain and/or express glycoproteins. In someembodiments, detection of an exogenous and/or non-native glycoproteinindicates the presence of microorganism in the cell composition. In someembodiments, a source of an exogenous and/or non-native glycan is aglycoprotein expressed or synthesized by cells, e.g., cells of a cellline, that are different from the cells of the cell composition. Incertain embodiments, detection of an exogenous and/or non-native glycansindicates cross contamination by other cell types and/or cell lines.

In some embodiments, the presence, identification, and/or quantificationof a non-native and/or exogenous glycan, e.g., by analyzing a surfaceglycan profile generated from a cell composition or a sample obtainedfrom the cell composition, corresponds to, correlates with, and/orindicates the presence, amount, and/or identity of one or moresubstances, e.g., residual substances, in the cell composition. In someembodiments, detection of a non-native and/or exogenous glycan indicatesa substance, e.g., a residual substance, within the cell composition. Insome embodiments, the non-native and/or exogenous glycan is thesubstance and/or residual substance. In certain embodiments, thesubstance and/or residual substance is a protein, e.g., a glycoprotein,that contains, e.g., is covalently bound to, the non-native and/orexogenous glycan. In particular embodiments, the substance is orincludes a microorganism or a substance produced from the microorganismthat expresses and/or synthesis, and/or is capable of expressing and/orsynthesizing, the non-native and/or exogenous glycan.

In some aspects, the level and/or amount of the substance, e.g., theresidual substance, present in the cell sample is determined bymeasuring the amount and/or level of the glycan, such as by any of themethods provided herein. In some embodiments, the amount or level of theglycan is quantified as a percentage of the total glycans in the sample.For example, in some embodiments, the methods provided herein allow fordetection of relative levels of an individual non-native or exogenousglycan species within a population of glycans. In particularembodiments, the area under one or more peaks corresponding to, e.g.,indicating, one or more non-native glycans of a liquid chromatograph canbe measured and expressed as a percentage of the total glycans detected.Such an analysis provides a relative percent amount of the exogenousand/or non-native glycan species within the total populations ofdetected glycans. In certain embodiments, a total amount orconcentration of the glycan may be calculated, such as for example, whenthe total amount of glycans are known.

In some embodiments, the glycan profile, e.g., the surface glycanprofile generated from a cell composition or from a sample obtained fromthe cell composition, is compared to a glycan profile of a referencesample. In some embodiments, the reference sample is or contains one ormore of a solution, media, and/or serum. In certain embodiments, thesolution, media, and/or serum are or have been contacted, treated,incubated, and/or exposed with the cells of the cell composition. Incertain embodiments, the reference sample is or includes a fresh sampleof the solution, media, and/or serum, e.g., the sample is taken from thesolution, media, or serum prior to exposure to the cells or is takenfrom a separate aliquot or container that has not been exposed to thecells. In particular embodiments, the presence of a glycan found in theglycan profile generated form the reference sample indicates thepresence of an exogenous and/or non-native glycan. In certainembodiments, the presence of an exogenous and/or non-native glycan in areference sample indicates that the source of the sample is, is likely,or is a candidate source of the substance and/or residual substance.

Particular embodiments contemplate that exogenous and/or non-nativeglycans may, in some instances, be incorporated into the cell surfaceglycans of one or more cells of the cell composition. In certain cases,an exogenous and/or non-native glycan is modified and/or altered, and isincorporated into the cell surface glycans of one or more cells. Thus,in some embodiments, a non-native and/or exogenous glycan that ispresent in a source, e.g., solution, media, and/or a serum, is modifiedwhen it contacts or is otherwise exposed to a cell composition. In suchembodiments, the identity of the exogenous and/or non-native glycan inthe source, e.g., the solution, media, and/or serum, is a differentexogenous and/or non-native glycan when present in the cell composition.Thus, in some instances, an exogenous or native glycan from a particularsource may be present in a surface glycan profile obtained from the cellcomposition, or from a sample of the cell composition, but not presentin the glycan profile generated from the reference sample taken from theparticular source. In some embodiments, control experiments can bedesigned and performed as a matter of routine to identify non-nativeand/or exogenous glycans that have been incorporated into the cellsurface glycans of a cell.

In some embodiments, the methods provided herein may be used to verifythat a substance has been rinsed, washed, or removed from the cells. Insome aspects, the methods provided herein may be used to compare cellculturing conditions and/or processes, e.g., engineering processes, tooptimize removal of the substances and/or residual substances that mayresult from the conditions or process, e.g., such as by residualproteins. In certain embodiments, surface glycan profiles are producedfrom samples taken at different stages of a process, e.g., anengineering process, to determine if the substances are introduced atone or more stages of the process.

II. CELL COMPOSITIONS

In some embodiments, the provided methods herein can be used to assessthe expression of surface glycans, e.g., N-glycans, of one or morecompositions of cells to determine or assess the presence, absence, orlevel of one or more glycans, e.g. N-glycans, present on the surface ofthe cells, such as according to any of the provided methods describedabove. In some embodiments, the composition that is assessed is a testcell composition that is a portion of another composition, such as asource composition. In some embodiments, the provided methods can beused to assess or analyze the presence, absence, amount, level and/orrelative abundance of one more glycans on the surface of cells of thecomposition.

In particular embodiments, such information can be used to assess orevaluate glycosylation changes that may impact functional and/orphenotypic characteristics of the cells, such as in connection with oneor more of masking of a cell surface marker, a metabolic activity,differentiation state, proliferative or expansion capacity, activationstate, cytolytic activity, signaling activity, an adhesion property, ora homing property. In some aspects, information about the glycan profilefrom cells of a composition can be compared to a reference sample, suchas a reference standard or other sample from a reference composition, toevaluate release criteria of the composition, evaluate changes ordifferences occurring in the composition, e.g. following incubation inthe presence of one or more agents or conditions and/or to evaluatesimilarities and/or differences between and among lots of the samecomposition produced by substantial the same method or to evaluatesimilarities or differences between and among compositions fromdifferent sources and/or produced or manufactured by different methods.

In some embodiments, information about a surface glycan profile, e.g.presence, absence, identity and/or level of one or more glycan, can beused in the process of screening one or more test agents or conditionson a composition of cells. In some embodiments, such methods includeincubating an input composition in the presence of one or more testagents or conditions as described. In some embodiments, the surfaceglycan profile, e.g. presence, absence, identity and/or level of one ormore surface glycans, from a sample from the composition of cells or aportion thereof, e.g. test cell composition, so incubated and comparedto a reference sample, such as a reference standard. In someembodiments, one or more test agent or conditions is selected forincubating or treating the cells if the comparison indicates the cellsurface glycan profile of the sample or each of the one or more targetglycan is substantially the same as the reference sample, e.g. referencestandard, and/or if the comparison indicates cell surface glycan profilecomprising the one or more target glycans or each of the one or moretarget glycans differs by no more than or about 20%, no more than orabout 15%, no more than or about 10% or no more than or about 5% fromthe cell surface glycan profile or each of the one or more targetglycans to the total glycans present in the reference sample.

In certain embodiments, the methods provided herein can be used toassess surface glycan expression, e.g. presence, absence, identityand/or level of one or more surface glycans, in a composition of cells,for example to compare the surface glycan expression of the compositionto another cell composition. In some embodiments, a sample containingone or more surface glycans released from a composition of cells, e.g.test cell composition, is compared to a reference sample and/or to asample of surface glycans released from a reference composition.

In particular embodiments, the presence, absence, relative amount (i.e.,percentage of total surface glycans), or amount of one or more targetglycan or target glycans can be assessed. In some embodiments, thetarget glycan or glycans can be species, families, or groups of glycans.In some embodiments, any of the glycans set forth in Table E1 can beassessed or compared. In some aspects, at least 50, at least 60, atleast 70, at least 80, at least 90, at least 100, or at least 200different species of glycans is assessed and/or compared. In someembodiments, the target glycan or glycans is or comprises high mannoseN-glycans, bisected and Sialyl Lewis^(X) N-glycans, and/or N-acetyllactosamine containing N-glycans. In some embodiments, the target glycanor glycans is or comprises a fucosylated biantennary complex glycanhaving no reducing end terminal galactose residues, a fucosylatedbiantennary complex glycan having one reducing end terminal galactoseresidue, a fucosylated biantennary complex glycan having two reducingend terminal galactose residues, a biantennary complex glycan having noreducing end terminal galactose residues, a biantennary complex glycanhaving one reducing end terminal galactose residue, a biantennarycomplex glycan having two reducing end terminal galactose residues, afucosylated biantennary complex glycan having two galactose residues andone N-acetylneuraminic acid residue, a fucosylated biantennary complexglycan having two galactose residues and two N-acetylneuraminic acidresidues, a biantennary complex glycan having two galactose residues andtwo N-acetylneuraminic acid residues, a high mannose glycan having fivemannose residues, a high mannose glycan having six mannose residues, ahigh mannose glycan having seven mannose residues, a high mannose glycanhaving eight mannose residues, and/or a high mannose glycan having ninemannose residues.

In some embodiments, the comparison of the surface glycan profile of acell composition may be compared to a composition of cells that expressa different recombinant receptor. In certain embodiments, the surfaceglycan composition may be compared to a composition of cells do notexpress the recombinant receptor. In some embodiments, the surfaceglycan composition may be compared to a composition of cells that isproduced by a different process and/or is produced by a processinvolving incubation in the presence of one or more particular agent orcondition. In some embodiments, the surface glycan profile of a cellcomposition is compared to a different cell composition from a differentstage or step of a manufacturing process for producing the cellcomposition, such an earlier or prior stage or step of the manufacturingprocess for producing the cell composition.

In certain embodiments, the surface glycan expression profile of a cellcomposition is assessed and compared to a reference sample. In someembodiments described herein, a reference sample is also referred to asa reference standard. For example, in some embodiments, a cellcomposition, e.g., a therapeutic cell composition may have a distinctprofile of surface glycan expression with respect to the level ofsurface expression of distinct glycan species, types of glycans, and/orglycan families as compared to compositions of different cells. Thus, insome embodiments, a reference standard may be generated by assessing aplurality of cell compositions. Such a reference standard may be used,in some embodiments, as a quality control and/or for a release assay, orto monitor or control the manufacture or culture of the cellcompositions.

In certain embodiments, a reference standard for surface glycanexpression is generated by assessing several different cell compositionsthat contain similar or identical cell compositions. For example, insome embodiments, surface glycan expression profiles are obtained fromtwo or more compositions of engineered cells that were manufacturedunder different batches of an identical manufacturing process togenerate a reference standard. In some embodiments, surface glycanexpression profiles are obtained from two or more compositions of cellsthat are collected at the same stage of a process to manufactureengineered cells to generate a reference standard. In some embodiments,the reference standard is a hypothetical reference standard value orrange of values with regard to specific glycan species. In someembodiments, a hypothetical reference standard is based upon data orreference samples from related or similar processes. In certain aspects,data from related processes can be shared such that reference valueranges can be generated that apply to myriad different, but similar,processes. In some embodiments, the reference standard is derived froman exemplary process to which other test processes are compared. In someembodiments, the reference standard is an average or median of thepresence, absence, identity and/or level of the one or more targetglycan or glycans among a plurality of compositions produced by theprocess.

In particular embodiments, the reference sample is generating bycalculating an average or median value of at least one species ofglycan, at least one family of N-glycan, or at least one type ofN-glycan. In certain embodiments, the reference standard is a median ormean level of expression of a type of glycan. In certain embodiments,the reference standard is or includes the mean or median level ofoligomannose, complex N-glycans, and/or hybrid N-glycans. In certainembodiments, the reference standard is or includes the mean or medianexpression level of N-glycans with high mannose content, bisected and/orsialyl Lewis^(X) N-glycans, and/or N-acetyl lactosamine containingN-glycans. In some embodiments, the reference standard is a mean ormedian expression level of the A2 family, the A2F family, the A3 family,the A4 family, and oligomannose family glycans. In some embodiments, thereference standard is a mean or median expression level of at least 1,2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100,125, 150, 200, 300, 400, or at least 500 glycan species. In someembodiments, the reference standard is calculated from the surfaceglycan expression profiles of at least two, three, four, five, six,seven, eight, nine, ten, greater than ten, greater than twenty, orgreater than fifty cell compositions.

A. Cells

In some embodiments, the cell composition, e.g. source composition or aportion thereof, such as a test cell composition, comprises a populationof cells. Any composition containing cells can be assessed according tothe provided method. In some embodiments, the population of cells is orcomprises a cell line or primary cells. In some embodiments, thepopulation of cells is or comprises primary cells, such as primary cellsobtained from a subject, e.g. human subject. In some embodiments, thepopulation of cells is or comprises stem cells, such as inducedpluripotent stem cells. In some embodiments, the composition of cells,e.g. source composition or a portion thereof, such as a test cellcomposition, is a composition that is associated with a process formanufacturing a cell composition, including in connection withengineering cells with a recombinant nucleic acid. In some embodiments,the composition of cells is a pharmaceutical composition.

In certain embodiments, the cells are or include eukaryotic cells. Incertain embodiments, the cells of the cell composition are animal cells.In some embodiments, the cells of the composition are mammalian cells.In certain embodiments, the cells are mouse cells, hamster cells, ratcells, or non-human primate cells. In some embodiments, the cells arehuman cells.

In some embodiments, the cells are cells of a cell line, e.g., e Chinesehamster ovary (CHO) cells, monkey kidney CV1 line transformed by 5V40(C057); human embryonic kidney line 293; baby hamster kidney cells(BHK); mouse sertoli cells (TM4); monkey kidney cells (CVI-76); Africangreen monkey kidney cells (VERO-76); human cervical carcinoma cells(HELA); canine kidney cells (MDCK); buffalo rat liver cells (BRL 3A);human lung cells (W138); human liver cells (Hep G2); mouse mammary tumorcells (MMT); rat hepatoma cells (HTC); HIH/3T3 cells, and TRI cells. Foran extensive list of mammalian cell lines, those of skill in the art mayrefer to the American Type Culture Collection catalog (ATCC, Mamassas,Va.). In some embodiments, the cells may be of a variety of cell types,e.g., fibroblasts, myoblasts, macrophages, or epithelial cells.

In particular embodiments, the cells of a composition are or includestem cells. In certain embodiments, cells of the cell composition arepluripotent stem cells, multipotent stem cells, oligopotent stem cells,and/or unipotent stem cells. In particular embodiments, the cells areinduced, e.g., induced pluripotent stem cells (ipsc). In particularembodiments, the cells of the composition are cells, e.g., that are inthe process of being reprogrammed, e.g., towards pluripotency. In someembodiments, the cells are stem cells are in the process ofdifferentiation.

In some embodiments, the cells of the composition are immune cells. Inparticular embodiments, a cell composition contains one or more of Tcells, B cells, and/or NK cells. In some embodiments the cells of thecell composition are CD3+ T cells. In some embodiments, the cells areCD4+ T cells. In certain embodiments, the cells are CD8+ T cells. Insome embodiments, one or more of effector T cells, Helper T cells,cytotoxic T cells, memory T cells, and suppresser T cells. In someembodiments, the cells are natural killer (NK) cells. In someembodiments, the cells are monocytes or granulocytes, e.g., myeloidcells, macrophages, neutrophils, dendritic cells, mast cells,eosinophils, and/or basophils.

In certain embodiments, the surface glycans of primary cells areassessed. In some embodiments, the composition of cells contains primarycells, such as those isolated directly from a subject and/or isolatedfrom a subject and frozen. In some embodiments, the cells include one ormore subsets of T cells or other cell types, such as whole T cellpopulations, CD4+ T cells, CD8+ T cells, and subpopulations thereof,such as those defined by function, activation state, maturity, potentialfor differentiation, expansion, recirculation, localization, and/orpersistence capacities, antigen-specificity, type of antigen receptor,presence in a particular organ or compartment, marker or cytokinesecretion profile, and/or degree of differentiation. With reference tothe subject to be treated, the cells may be allogeneic and/orautologous.

In some embodiments, one or more cells of the composition are engineeredcells. In some cases, the one or more cells are engineered to contain arecombinant nucleic acid, e.g. contain heterologous nucleic acid and/orexpress a heterologous protein. In some embodiments, the recombinantnucleic acid encodes a recombinant protein. In some cases, therecombinant protein can be any protein that is desired to be expressedor produced by a recombinant cell composition. In some embodiments, therecombinant protein is a recombinant receptor. In some embodiments, therecombinant nucleic acid is or includes a viral vector, e.g. lentiviralor retroviral vector, that is transferred or introduced into the cellfor expression of the recombinant protein.

B. Exemplary Engineered Cells

In certain embodiments, the engineered cells contain a heterologouspolynucleotide that encodes a recombinant receptor. In some embodiments,the recombinant receptor is a chimeric receptor or an antigen receptor,such as a chimeric antigen receptor (CAR) or a T cell receptor (TCR). Incertain embodiments, the engineered cells are produced, manufactured,and/or generated as described in Section III.

In some embodiments, all or a portion of the cells in a compositioncontain or are engineered to contain an engineered receptor, such as achimeric antigen receptor (CAR), or a T cell receptor (TCR). Inparticular embodiments, all or a portion of the cells in a compositionexpress the engineered receptor. In some embodiments, compositionscontaining engineered cells are enriched for such cells. In certainembodiments, the cells of a certain type such as T cells or CD8⁺ or CD4T cells are enriched or selected. In particular embodiments, the cellcomposition is a therapeutic and/or a pharmaceutical cell composition,such as for adoptive cell therapy.

1. Chimeric Antigen Receptors (CARs)

In certain embodiments, the methods provided herein can be used toassess the surface glycan expression of a composition of cells thatincludes or is composed of cells that generally express recombinantreceptors, such as antigen receptors including functional non-TCRantigen receptors, e.g., chimeric antigen receptors (CARs), and otherantigen-binding receptors such as transgenic T cell receptors (TCRs).Also among the receptors are other chimeric receptors.

Exemplary antigen receptors, including CARs, and methods for engineeringand introducing such receptors into cells, include those described, forexample, in international patent application publication numbersWO200014257, WO2013126726, WO2012/129514, WO2014031687, WO2013/166321,WO2013/071154, WO2013/123061 U.S. patent application publication numbersUS2002131960, US2013287748, US20130149337, U.S. Pat. Nos. 6,451,995,7,446,190, 8,252,592, 8,339,645, 8,398,282, 7,446,179, 6,410,319,7,070,995, 7,265,209, 7,354,762, 7,446,191, 8,324,353, and 8,479,118,and European patent application number EP2537416, and/or those describedby Sadelain et al., Cancer Discov., 3(4): 388-398 (2013); Davila et al.PLoS ONE 8(4): e61338 (2013); Turtle et al., Curr. Opin. Immunol.,24(5): 633-39 (2012); Wu et al., Cancer, 18(2): 160-75 (2012). In someaspects, the antigen receptors include a CAR as described in U.S. Pat.No. 7,446,190, and those described in International Patent ApplicationPublication No.: WO/2014055668 A1. Examples of the CARs include CARs asdisclosed in any of the aforementioned publications, such asWO2014031687, U.S. Pat. Nos. 8,339,645, 7,446,179, US 2013/0149337, U.S.Pat. Nos. 7,446,190, 8,389,282, Kochenderfer et al., Nature ReviewsClinical Oncology, 10, 267-276 (2013); Wang et al., I Immunother. 35(9):689-701 (2012); and Brentjens et al., Sci Transl Med,. 5(177) (2013).See also WO2014031687, U.S. Pat. Nos. 8,339,645, 7,446,179, US2013/0149337, U.S. Pat. Nos. 7,446,190, and 8,389,282. The chimericreceptors, such as CARs, generally include an extracellular antigenbinding domain, such as a portion of an antibody molecule, generally avariable heavy (VH) chain region and/or variable light (VL) chain regionof the antibody, e.g., an scFv antibody fragment.

In some embodiments, surface expression of N-glycans is assessed in acomposition of cells that contains cells expressing a recombinantreceptor, e.g., a CAR, that targets an antigen, and the antigen targetedby the receptor is a polypeptide. In some embodiments, the antigen is acarbohydrate or other molecule. In some embodiments, the antigen isselectively expressed or overexpressed on cells of a disease orcondition, e.g., tumor cells or pathogenic cells, as compared to normalor non-targeted cells or tissues. In other embodiments, the antigen isexpressed on normal cells and/or is expressed on the engineered cells.In some embodiments, among the antigens targeted by the chimericreceptors are those expressed in the context of a disease, condition, orcell type to be targeted via the adoptive cell the In some embodiments,the labeling reagent and/or derivatizing reagent is contacted, treated,and/or incubated with the glycans in the presence of a solvent that is aformamide. Among the diseases and conditions are proliferative,neoplastic, and malignant diseases and disorders, including cancers andtumors, including hematologic cancers, cancers of the immune system,such as lymphomas, leukemia, and/or myelomas, such as B, T, and myeloidleukemia, lymphomas, and multiple myelomas.

In some embodiments, surface glycans are released from a cellcomposition containing cells that express a recombinant receptor, e.g.,a CAR, that binds to an antigen. Antigens that may be targeted by thereceptors include, but are not limited to, αvβ6 integrin (avb6integrin), B cell maturation antigen (BCMA), B7-H3, B7-H6, carbonicanhydrase 9 (CA9, also known as CAIX or G250), a cancer-testis antigen,cancer/testis antigen 1B (CTAG, also known as NY-ESO-1 and LAGE-2),carcinoembryonic antigen (CEA), a cyclin, cyclin A2, C-C Motif ChemokineLigand 1 (CCL-1), CD19, CD20, CD22, CD23, CD24, CD30, CD33, CD38, CD44,CD44v6, CD44v7/8, CD123, CD133, CD138, CD171, chondroitin sulfateproteoglycan 4 (CSPG4), epidermal growth factor protein (EGFR),truncated epidermal growth factor protein (tEGFR), type III epidermalgrowth factor receptor mutation (EGFR vIII), epithelial glycoprotein 2(EPG-2), epithelial glycoprotein 40 (EPG-40), ephrinB2, ephrine receptorA2 (EPHa2), estrogen receptor, Fc receptor like 5 (FCRLS; also known asFc receptor homolog 5 or FCRHS), fetal acetylcholine receptor (fetalAchR), a folate binding protein (FBP), folate receptor alpha, fetalacetylcholine receptor, ganglioside GD2, O-acetylated GD2 (OGD2),ganglioside GD3, glycoprotein 100 (gp100), glypican-3 (GPC3), Her2/neu(receptor tyrosine kinase erbB2), Her3 (erb-B3), Her4 (erb-B4), erbBdimers, human high molecular weight-melanoma-associated antigen(HMW-MAA), hepatitis B surface antigen, Human leukocyte antigen A1(HLA-A1), human leukocyte antigen A2 (HLA-A2), IL-22 receptoralpha(IL-22Rα), IL-13 receptor alpha 2 (IL-13Rα2), kinase insert domainreceptor (kdr), kappa light chain, L1 cell adhesion molecule (L1CAM),CE7 epitope of L1-CAM, Leucine Rich Repeat Containing 8 Family Member A(LRRC8A), Lewis Y, melanoma-associated antigen (MAGE)-A1, MAGE-A3,MAGE-A6, MAGE-A10, mesothelin (MSLN), c-Met, murine cytomegalovirus(CMV), mucin 1 (MUC1), MUC16, natural killer group 2 member D (NKG2D)ligands, melan A (MART-1), neural cell adhesion molecule (NCAM),oncofetal antigen, preferentially expressed antigen of melanoma (PRAME),progesterone receptor, a prostate specific antigen, prostate stem cellantigen (PSCA), prostate specific membrane antigen (PSMA), receptortyrosine kinase like orphan receptor 1 (ROR1), survivin, Trophoblastglycoprotein (TPBG also known as 5T4), tumor-associated glycoprotein 72(TAG72), Tyrosinase related protein 1 (TRP1, also known as TYRP1 orgp75), Tyrosinase related protein 2 (TRP2, also known as dopachrometautomerase, dopachrome delta-isomerase or DCT), vascular endothelialgrowth factor receptor (VEGFR), vascular endothelial growth factorreceptor 2 (VEGFR2), Wilms tumor 1 (WT-1), and a pathogen-specific orpathogen-expressed antigen, and/or molecules expressed by HIV, HCV, HBVor other pathogens. Antigens targeted by the receptors in someembodiments include antigens associated with a B cell malignancy, suchas any of a number of known B cell marker. In some embodiments, theantigen is or includes CD20, CD19, CD22, ROR1, CD45, CD21, CD5, CD33,Igkappa, Iglambda, CD79a, CD79b or CD30.

In some embodiments, the antigen is a pathogen-specific or pathogenexpressed antigen. In some embodiments, the CAR is specific for viralantigens (such as HIV, HCV, HBV, etc.), bacterial antigens, and/orparasitic antigens.

In some embodiments, the antibody or an antigen-binding fragment (e.g.scFv or VII domain) specifically recognizes an antigen, such as CD19. Insome embodiments, the antibody or antigen-binding fragment is derivedfrom, or is a variant of, antibodies or antigen-binding fragment thatspecifically binds to CD19.

In some embodiments the scFv and/or VII domains is derived from FMC63.FMC63 generally refers to a mouse monoclonal IgG1 antibody raisedagainst Nalm-1 and -16 cells expressing CD19 of human origin (Ling, N.R., et al. (1987). Leucocyte typing III. 302). The FMC63 antibodycomprises CDR H1 set forth in SEQ ID NO: 29; CDR H2 set forth in SEQ IDNO: 30; CDR H3 set forth in SEQ ID NOS: 31 or 45; and CDR L1 set forthin SEQ ID NO: 26; CDR L2 set forth in SEQ ID NO: 27 or 46; and CDR L3set forth in SEQ ID NO: 28 or 45. The FMC63 antibody comprises the heavychain variable region (VII) comprising the amino acid sequence of SEQ IDNO: 32 and the light chain variable region (VL) comprising the aminoacid sequence of SEQ ID NO: 33. In some embodiments, the scFv comprisesa variable light chain containing a CDR L1 sequence of SEQ ID NO: 26, aCDR L2 sequence of SEQ ID NO: 28, and a CDR L3 sequence of SEQ ID NO: 28and/or a variable heavy chain containing a CDR H1 sequence of SEQ ID NO:29, a CDR H2 sequence of SEQ ID NO: 30, and a CDR H3 sequence of SEQ IDNO: 31, or a variant of any of the foregoing having at least 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or moresequence identity thereto. In some embodiments, the scFv comprises avariable heavy chain region of FMC63 set forth in SEQ ID NO: 32 and avariable light chain region of FMC63 set forth in SEQ ID NO: 33, or avariant of any of the foregoing having at least 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequenceidentity thereto. In some embodiments, the variable heavy and variablelight chains are connected by a linker. In some embodiments, the linkeris set forth in SEQ ID NO:58. In some embodiments, the scFv comprises,in order, a V_(H), a linker, and a VL. In some embodiments, the scFvcomprises, in order, a V_(L), a linker, and a V_(H). In someembodiments, the scFv is encoded by a sequence of nucleotides set forthin SEQ ID NO: 25 or a sequence that exhibits at least 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequenceidentity to SEQ ID NO: 25. In some embodiments, the scFv comprises thesequence of amino acids set forth in SEQ ID NO: 34 or a sequence thatexhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 34.

In some embodiments, the scFv and/or VII domain is derived from SJ25C1.SJ25C1 is a mouse monoclonal IgG1 antibody raised against Nalm-1 and -16cells expressing CD19 of human origin (Ling, N. R., et al. (1987).Leucocyte typing III. 302). The SJ25C1 antibody comprises CDR H1, H2 andH3 set forth in SEQ ID NOS: 38-40, respectively, and CDR L1, L2 and L3sequences set forth in SEQ ID NOS: 35-37, respectively. The SJ25C1antibody comprises the heavy chain variable region (V_(H)) comprisingthe amino acid sequence of SEQ ID NO: 41 and the light chain variableregion (V_(L)) comprising the amino acid sequence of SEQ ID NO: 42. Insome embodiments, the svFv comprises a variable light chain containing aCDR L1 sequence set forth in SEQ ID NO:35; a CDR L2 set forth in SEQ IDNO: 36; and a CDR L3 set forth in SEQ ID NO:37; and/or a variable heavychain containing a CDR H1 set forth in SEQ ID NO:38, a CDR H2 set forthin SEQ ID NO:39, and a CDR H3 set forth in SEQ ID NO:40, or a variant ofany of the foregoing having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identitythereto. In some embodiments, the scFv comprises a variable heavy chainregion of SJ25C1 set forth in SEQ ID NO:41 and a variable light chainregion of SJ25C1 set forth in SEQ ID NO: 42, or a variant of any of theforegoing having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto. In someembodiments, the variable heavy and variable light chains are connectedby a linker. In some embodiments, the linker is set forth in SEQ ID NO:43. In some embodiments, the scFv comprises, in order, a VII, a linker,and a V_(L). In some embodiments, the scFv comprises, in order, a V_(L),a linker, and a VII. In some embodiments, the scFv comprises thesequence of amino acids set forth in SEQ ID NO:44 or a sequence thatexhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:44.

In some aspects, the CAR contains a ligand- (e.g., antigen-) bindingdomain that binds or recognizes, e.g., specifically binds, a universaltag or a universal epitope. In some aspects, the binding domain can binda molecule, a tag, a polypeptide and/or an epitope that can be linked toa different binding molecule (e.g., antibody or antigen-bindingfragment) that recognizes an antigen associated with a disease ordisorder. Exemplary tag or epitope includes a dye (e.g., fluoresceinisothiocyanate) or a biotin. In some aspects, a binding molecule (e.g.,antibody or antigen-binding fragment) linked to a tag, that recognizesthe antigen associated with a disease or disorder, e.g., tumor antigen,with an engineered cell expressing a CAR specific for the tag, to effectcytotoxicity or other effector function of the engineered cell. In someaspects, the specificity of the CAR to the antigen associated with adisease or disorder is provided by the tagged binding molecule (e.g.,antibody), and different tagged binding molecule can be used to targetdifferent antigens. Exemplary CARs specific for a universal tag or auniversal epitope include those described, e.g., in U.S. Pat. No.9,233,125, WO 2016/030414, Urbanska et al., (2012) Cancer Res 72:1844-1852, and Tamada et al., (2012). Clin Cancer Res 18:6436-6445.

In some embodiments, the CAR contains a TCR-like antibody, such as anantibody or an antigen-binding fragment (e.g. scFv) that specificallyrecognizes an intracellular antigen, such as a tumor-associated antigen,presented on the cell surface as a major histocompatibility complex(MHC)-peptide complex. In some embodiments, an antibody orantigen-binding portion thereof that recognizes an MHC-peptide complexcan be expressed on cells as part of a recombinant receptor, such as anantigen receptor. Among the antigen receptors are functional non-T cellreceptor (TCR) antigen receptors, such as chimeric antigen receptors(CARs). In some embodiments, a CAR containing an antibody orantigen-binding fragment that exhibits TCR-like specificity directedagainst peptide-MHC complexes also may be referred to as a TCR-like CAR.In some embodiments, the CAR is a TCR-like CAR and the antigen is aprocessed peptide antigen, such as a peptide antigen of an intracellularprotein, which, like a TCR, is recognized on the cell surface in thecontext of an MHC molecule. In some embodiments, the extracellularantigen-binding domain specific for an MHC-peptide complex of a TCR-likeCAR is linked to one or more intracellular signaling components, in someaspects via linkers and/or transmembrane domain(s). In some embodiments,such molecules can typically mimic or approximate a signal through anatural antigen receptor, such as a TCR, and, optionally, a signalthrough such a receptor in combination with a costimulatory receptor.

Reference to “Major histocompatibility complex” (MHC) refers to aprotein, generally a glycoprotein, that contains a polymorphic peptidebinding site or binding groove that can, in some cases, complex withpeptide antigens of polypeptides, including peptide antigens processedby the cell machinery. In some cases, MHC molecules can be displayed orexpressed on the cell surface, including as a complex with peptide, i.e.MHC-peptide complex, for presentation of an antigen in a conformationrecognizable by an antigen receptor on T cells, such as a TCRs orTCR-like antibody. Generally, MHC class I molecules are heterodimershaving a membrane spanning α chain, in some cases with three α domains,and a non-covalently associated β2 microglobulin. Generally, MHC classII molecules are composed of two transmembrane glycoproteins, α and β,both of which typically span the membrane. An MHC molecule can includean effective portion of an MHC that contains an antigen binding site orsites for binding a peptide and the sequences necessary for recognitionby the appropriate antigen receptor. In some embodiments, MHC class Imolecules deliver peptides originating in the cytosol to the cellsurface, where a MHC-peptide complex is recognized by T cells, such asgenerally CD8+ T cells, but in some cases CD4+ T cells. In someembodiments, MHC class II molecules deliver peptides originating in thevesicular system to the cell surface, where they are typicallyrecognized by CD4+ T cells. Generally, MHC molecules are encoded by agroup of linked loci, which are collectively termed H-2 in the mouse andhuman leukocyte antigen (HLA) in humans. Hence, typically human MHC canalso be referred to as human leukocyte antigen (HLA).

The term “MHC-peptide complex” or “peptide-MHC complex” or variationsthereof, refers to a complex or association of a peptide antigen and anMHC molecule, such as, generally, by non-covalent interactions of thepeptide in the binding groove or cleft of the MHC molecule. In someembodiments, the MHC-peptide complex is present or displayed on thesurface of cells. In some embodiments, the MHC-peptide complex can bespecifically recognized by an antigen receptor, such as a TCR, TCR-likeCAR or antigen-binding portions thereof.

In some embodiments, a peptide, such as a peptide antigen or epitope, ofa polypeptide can associate with an MHC molecule, such as forrecognition by an antigen receptor. Generally, the peptide is derivedfrom or based on a fragment of a longer biological molecule, such as apolypeptide or protein. In some embodiments, the peptide typically isabout 8 to about 24 amino acids in length. In some embodiments, apeptide has a length of from or from about 9 to 22 amino acids forrecognition in the MHC Class II complex. In some embodiments, a peptidehas a length of from or from about 8 to 13 amino acids for recognitionin the MHC Class I complex. In some embodiments, upon recognition of thepeptide in the context of an MHC molecule, such as MHC-peptide complex,the antigen receptor, such as TCR or TCR-like CAR, produces or triggersan activation signal to the T cell that induces a T cell response, suchas T cell proliferation, cytokine production, a cytotoxic T cellresponse or other response.

In some embodiments, a TCR-like antibody or antigen-binding portion, areknown or can be produced by known methods (see e.g. US PublishedApplication Nos. US 2002/0150914; US 2003/0223994; US 2004/0191260; US2006/0034850; US 2007/00992530; US20090226474; US20090304679; andInternational App. Pub. No. WO 03/068201).

In some embodiments, an antibody or antigen-binding portion thereof thatspecifically binds to a MHC-peptide complex, can be produced byimmunizing a host with an effective amount of an immunogen containing aspecific MHC-peptide complex. In some cases, the peptide of theMHC-peptide complex is an epitope of antigen capable of binding to theMHC, such as a tumor antigen, for example a universal tumor antigen,myeloma antigen or other antigen as described below. In someembodiments, an effective amount of the immunogen is then administeredto a host for eliciting an immune response, wherein the immunogenretains a three-dimensional form thereof for a period of time sufficientto elicit an immune response against the three-dimensional presentationof the peptide in the binding groove of the MHC molecule. Serumcollected from the host is then assayed to determine if desiredantibodies that recognize a three-dimensional presentation of thepeptide in the binding groove of the MHC molecule is being produced. Insome embodiments, the produced antibodies can be assessed to confirmthat the antibody can differentiate the MHC-peptide complex from the MHCmolecule alone, the peptide of interest alone, and a complex of MHC andirrelevant peptide. The desired antibodies can then be isolated.

In some embodiments, an antibody or antigen-binding portion thereof thatspecifically binds to an MHC-peptide complex can be produced byemploying antibody library display methods, such as phage antibodylibraries. In some embodiments, phage display libraries of mutant Fab,scFv or other antibody forms can be generated, for example, in whichmembers of the library are mutated at one or more residues of a CDR orCDRs. See e.g. US Pat. App. Pub. No. US20020150914, US20140294841; andCohen CJ. et al. (2003) J Mol. Recogn. 16:324-332.

[0041][0037] The term “antibody” herein is used in the broadest senseand includes polyclonal and monoclonal antibodies, including intactantibodies and functional (antigen-binding) antibody fragments,including fragment antigen binding (Fab) fragments, F(ab′)2 fragments,Fab′ fragments, Fv fragments, recombinant IgG (rIgG) fragments, variableheavy chain (V_(H)) regions capable of specifically binding the antigen,single chain antibody fragments, including single chain variablefragments (scFv), and single domain antibodies (e.g., sdAb, sdFv,nanobody, VHH or VNAR) or fragments. The term encompasses geneticallyengineered and/or otherwise modified forms of immunoglobulins, such asintrabodies, peptibodies, chimeric antibodies, fully human antibodies,humanized antibodies, and heteroconjugate antibodies, multispecific,e.g., bispecific, antibodies, diabodies, triabodies, and tetrabodies,tandem di-scFv, tandem tri-scFv. Unless otherwise stated, the term“antibody” should be understood to encompass functional antibodyfragments thereof. The term also encompasses intact or full-lengthantibodies, including antibodies of any class or sub-class, includingIgG and sub-classes thereof, IgM, IgE, IgA, and IgD. In some aspects,the CAR is a bispecific CAR, e.g., containing two antigen-bindingdomains with different specificities.

In some embodiments, the antigen-binding proteins, antibodies andantigen binding fragments thereof specifically recognize an antigen of afull-length antibody. In some embodiments, the heavy and light chains ofan antibody can be full-length or can be an antigen-binding portion (aFab, F(ab′)2, Fv or a single chain Fv fragment (scFv)). In otherembodiments, the antibody heavy chain constant region is chosen from,e.g., IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE,particularly chosen from, e.g., IgG1, IgG2, IgG3, and IgG4, moreparticularly, IgG1 (e.g., human IgG1). In another embodiment, theantibody light chain constant region is chosen from, e.g., kappa orlambda, particularly kappa.

Among the provided antibodies are antibody fragments. An “antibodyfragment” refers to a molecule other than an intact antibody thatcomprises a portion of an intact antibody that binds the antigen towhich the intact antibody binds. Examples of antibody fragments includebut are not limited to Fv, Fab, Fab′, Fab′-SH, F(ab′)2; diabodies;linear antibodies; variable heavy chain (VH) regions, single-chainantibody molecules such as scFvs and single-domain VH single antibodies;and multispecific antibodies formed from antibody fragments. Inparticular embodiments, the antibodies are single-chain antibodyfragments comprising a variable heavy chain region and/or a variablelight chain region, such as scFvs.

The term “variable region” or “variable domain” refers to the domain ofan antibody heavy or light chain that is involved in binding theantibody to antigen. The variable domains of the heavy chain and lightchain (VH and VL, respectively) of a native antibody generally havesimilar structures, with each domain comprising four conserved frameworkregions (FRs) and three CDRs. (See, e.g., Kindt et al. Kuby Immunology,6th ed., W.H. Freeman and Co., page 91 (2007). A single VH or VL domainmay be sufficient to confer antigen-binding specificity. Furthermore,antibodies that bind a particular antigen may be isolated using a VH orVL domain from an antibody that binds the antigen to screen a library ofcomplementary VL or VH domains, respectively. See, e.g., Portolano etal., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628(1991).

Single-domain antibodies (sdAb) are antibody fragments comprising all ora portion of the heavy chain variable domain or all or a portion of thelight chain variable domain of an antibody. In certain embodiments, asingle-domain antibody is a human single-domain antibody. In someembodiments, the CAR comprises an antibody heavy chain domain thatspecifically binds the antigen, such as a cancer marker or cell surfaceantigen of a cell or disease to be targeted, such as a tumor cell or acancer cell, such as any of the target antigens described herein orknown. Exemplary single-domain antibodies include sdFv, nanobody, VHH orVNAR.

Antibody fragments can be made by various techniques, including but notlimited to proteolytic digestion of an intact antibody as well asproduction by recombinant host cells. In some embodiments, theantibodies are recombinantly produced fragments, such as fragmentscomprising arrangements that do not occur naturally, such as those withtwo or more antibody regions or chains joined by synthetic linkers,e.g., peptide linkers, and/or that are may not be produced by enzymedigestion of a naturally-occurring intact antibody. In some embodiments,the antibody fragments are scFvs.

A “humanized” antibody is an antibody in which all or substantially allCDR amino acid residues are derived from non-human CDRs and all orsubstantially all FR amino acid residues are derived from human FRs. Ahumanized antibody optionally may include at least a portion of anantibody constant region derived from a human antibody. A “humanizedform” of a non-human antibody, refers to a variant of the non-humanantibody that has undergone humanization, typically to reduceimmunogenicity to humans, while retaining the specificity and affinityof the parental non-human antibody. In some embodiments, some FRresidues in a humanized antibody are substituted with correspondingresidues from a non-human antibody (e.g., the antibody from which theCDR residues are derived), e.g., to restore or improve antibodyspecificity or affinity.

In some embodiments, the antibody portion of the recombinant receptor,e.g., CAR, further includes at least a portion of an immunoglobulinconstant region, such as a hinge region, e.g., an IgG4 hinge region,and/or a CH1/CL and/or Fc region. In some embodiments, the constantregion or portion is of a human IgG, such as IgG4 or IgG1. In someaspects, the portion of the constant region serves as a spacer regionbetween the antigen-recognition component, e.g., scFv, and transmembranedomain. The spacer can be of a length that provides for increasedresponsiveness of the cell following antigen binding, as compared to inthe absence of the spacer. Exemplary spacers, e.g., hinge regions,include those described in international patent application publicationnumber WO2014031687. In some examples, the spacer is or is about 12amino acids in length or is no more than 12 amino acids in length.Exemplary spacers include those having at least about 10 to 229 aminoacids, about 10 to 200 amino acids, about 10 to 175 amino acids, about10 to 150 amino acids, about 10 to 125 amino acids, about 10 to 100amino acids, about 10 to 75 amino acids, about 10 to 50 amino acids,about 10 to 40 amino acids, about 10 to 30 amino acids, about 10 to 20amino acids, or about 10 to 15 amino acids, and including any integerbetween the endpoints of any of the listed ranges. In some embodiments,the spacer is less than 250 amino acids in length, less than 200 aminoacids in length, less than 150 amino acids in length, less than 100amino acids in length, less than 75 amino acids in length, less than 50amino acids in length, less than 25 amino acids in length, less than 20amino acids in length, less than 15 amino acids in length, less than 12amino acids in length, or less than 10 amino acids in length. In someembodiments, the spacer is from or from about 10 to 250 amino acids inlength, 10 to 150 amino acids in length, 10 to 100 amino acids inlength, 10 to 50 amino acids in length, 10 to 25 amino acids in length,10 to 15 amino acids in length, 15 to 250 amino acids in length, 15 to150 amino acids in length, 15 to 100 amino acids in length, 15 to 50amino acids in length, 15 to 25 amino acids in length, 25 to 250 aminoacids in length, 25 to 100 amino acids in length, 25 to 50 amino acidsin length, 50 to 250 amino acids in length, 50 to 150 amino acids inlength, 50 to 100 amino acids in length, 100 to 250 amino acids inlength, 100 to 150 amino acids in length, or 150 to 250 amino acids inlength. In some embodiments, a spacer region has about 12 amino acids orless, about 119 amino acids or less, or about 229 amino acids or less.Exemplary spacers include IgG4 hinge alone, IgG4 hinge linked to CH2 andCH3 domains, or IgG4 hinge linked to the CH3 domain. Exemplary spacersinclude, but are not limited to, those described in Hudecek et al. Clin.Cancer Res., 19:3153 (2013), international patent applicationpublication number WO2014031687, U.S. Pat. No. 8,822,647 or publishedapp. No. US2014/0271635.

In some embodiments, the constant region or portion is of a human IgG,such as IgG4 or IgG1. In some embodiments, the spacer has the sequenceESKYGPPCPPCP (set forth in SEQ ID NO: 2), and is encoded by the sequenceset forth in SEQ ID NO: 3. In some embodiments, the spacer has thesequence set forth in SEQ ID NO: 4. In some embodiments, the spacer hasthe sequence set forth in SEQ ID NO: 5. In some embodiments, theconstant region or portion is of IgD. In some embodiments, the spacerhas the sequence set forth in SEQ ID NO: 6. In some embodiments, thespacer has a sequence of amino acids that exhibits at least 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or moresequence identity to any of SEQ ID NOS: 2, 4, 5, or 6.

This antigen recognition domain generally is linked to one or moreintracellular signaling components, such as signaling components thatmimic activation through an antigen receptor complex, such as a TCRcomplex, in the case of a CAR, and/or signal via another cell surfacereceptor. Thus, in some embodiments, the antigen-binding component(e.g., antibody) is linked to one or more transmembrane andintracellular signaling domains. In some embodiments, the transmembranedomain is fused to the extracellular domain. In one embodiment, atransmembrane domain that naturally is associated with one of thedomains in the receptor, e.g., CAR, is used. In some instances, thetransmembrane domain is selected or modified by amino acid substitutionto avoid binding of such domains to the transmembrane domains of thesame or different surface membrane proteins to minimize interactionswith other members of the receptor complex.

The transmembrane domain in some embodiments is derived either from anatural or from a synthetic source. Where the source is natural, thedomain in some aspects is derived from any membrane-bound ortransmembrane protein. Transmembrane regions include those derived from(i.e. comprise at least the transmembrane region(s) of) the alpha, betaor zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5,CDS, CD9, CD 16, CD22, CD33, CD37, CD64, CD80, CD86, CD 134, CD137, CD154. Alternatively the transmembrane domain in some embodiments issynthetic. In some aspects, the synthetic transmembrane domain comprisespredominantly hydrophobic residues such as leucine and valine. In someaspects, a triplet of phenylalanine, tryptophan and valine will be foundat each end of a synthetic transmembrane domain. In some embodiments,the linkage is by linkers, spacers, and/or transmembrane domain(s).

Among the intracellular signaling domains are those that mimic orapproximate a signal through a natural antigen receptor, a signalthrough such a receptor in combination with a costimulatory receptor,and/or a signal through a costimulatory receptor alone. In someembodiments, a short oligo- or polypeptide linker, for example, a linkerof between 2 and 10 amino acids in length, such as one containingglycines and serines, e.g., glycine-serine doublet, is present and formsa linkage between the transmembrane domain and the cytoplasmic signalingdomain of the CAR.

The receptor, e.g., the CAR, generally includes at least oneintracellular signaling component or components. In some embodiments,the receptor includes an intracellular component of a TCR complex, suchas a TCR CD3 chain that mediates T-cell activation and cytotoxicity,e.g., CD3 zeta chain. Thus, in some aspects, the antigen-binding portionis linked to one or more cell signaling modules. In some embodiments,cell signaling modules include CD3 transmembrane domain, CD3intracellular signaling domains, and/or other CD transmembrane domains.In some embodiments, the receptor, e.g., CAR, further includes a portionof one or more additional molecules such as Fc receptor γ, CD8, CD4,CD25, or CD16. For example, in some aspects, the CAR or other chimericreceptor includes a chimeric molecule between CD3-zeta (CD3-ζ) or Fcreceptor γ and CD8, CD4, CD25 or CD16.

In some embodiments, upon ligation of the CAR or other chimericreceptor, the cytoplasmic domain or intracellular signaling domain ofthe receptor activates at least one of the normal effector functions orresponses of the immune cell, e.g., T cell engineered to express theCAR. For example, in some contexts, the CAR induces a function of a Tcell such as cytolytic activity or T-helper activity, such as secretionof cytokines or other factors. In some embodiments, a truncated portionof an intracellular signaling domain of an antigen receptor component orcostimulatory molecule is used in place of an intact immunostimulatorychain, for example, if it transduces the effector function signal. Insome embodiments, the intracellular signaling domain or domains includethe cytoplasmic sequences of the T cell receptor (TCR), and in someaspects also those of co-receptors that in the natural context act inconcert with such receptors to initiate signal transduction followingantigen receptor engagement.

In certain embodiments, methods provided engineered cells that expressone or more recombinant antigen receptor. In some embodiments, the cellscan include cells genetically engineered with a recombinant receptor,such as a chimeric antigen receptor.

In the context of a natural TCR, full activation generally requires notonly signaling through the TCR, but also a costimulatory signal. Thus,in some embodiments, to promote full activation, a component forgenerating secondary or co-stimulatory signal is also included in theCAR. In other embodiments, the CAR does not include a component forgenerating a costimulatory signal. In some aspects, an additional CAR isexpressed in the same cell and provides the component for generating thesecondary or costimulatory signal.

T cell activation is in some aspects described as being mediated by twoclasses of cytoplasmic signaling sequences: those that initiateantigen-dependent primary activation through the TCR (primarycytoplasmic signaling sequences), and those that act in anantigen-independent manner to provide a secondary or co-stimulatorysignal (secondary cytoplasmic signaling sequences). In some aspects, theCAR includes one or both of such signaling components.

In some aspects, the CAR includes a primary cytoplasmic signalingsequence that regulates primary activation of the TCR complex. Primarycytoplasmic signaling sequences that act in a stimulatory manner maycontain signaling motifs which are known as immunoreceptortyrosine-based activation motifs or ITAMs. Examples of ITAM containingprimary cytoplasmic signaling sequences include those derived from TCRzeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CDS, CD22,CD79a, CD79b, and CD66d. In some embodiments, cytoplasmic signalingmolecule(s) in the CAR contain(s) a cytoplasmic signaling domain,portion thereof, or sequence derived from CD3 zeta.

In some embodiments, the CAR includes a signaling domain and/ortransmembrane portion of a costimulatory receptor, such as CD28, 4-1BB,OX40, DAP10, and ICOS. In some aspects, the same CAR includes both theactivating and costimulatory components.

In some embodiments, the activating domain is included within one CAR,whereas the costimulatory component is provided by another CARrecognizing another antigen. In some embodiments, the CARs includeactivating or stimulatory CARs, costimulatory CARs, both expressed onthe same cell (see WO2014/055668). In some aspects, the cells includeone or more stimulatory or activating CAR and/or a costimulatory CAR. Insome embodiments, the cells further include inhibitory CARs (iCARs, seeFedorov et al., Sci. Transl. Medicine, 5(215) (2013), such as a CARrecognizing an antigen other than the one associated with and/orspecific for the disease or condition whereby an activating signaldelivered through the disease-targeting CAR is diminished or inhibitedby binding of the inhibitory CAR to its ligand, e.g., to reduceoff-target effects.

In certain embodiments, the intracellular signaling domain comprises aCD28 transmembrane and signaling domain linked to a CD3 (e.g., CD3-zeta)intracellular domain. In some embodiments, the intracellular signalingdomain comprises a chimeric CD28 and CD137 (4-1BB, TNFRSF9)co-stimulatory domains, linked to a CD3 zeta intracellular domain.

In some embodiments, the CAR encompasses one or more, e.g., two or more,costimulatory domains and an activation domain, e.g., primary activationdomain, in the cytoplasmic portion. Exemplary CARs include intracellularcomponents of CD3-zeta, CD28, and 4-1BB.

In some embodiments, the CAR or other antigen receptor further includesa marker, such as a cell surface marker, which may be used to confirmtransduction or engineering of the cell to express the receptor, such asa truncated version of a cell surface receptor, such as truncated EGFR(tEGFR). In some aspects, the marker includes all or part (e.g.,truncated form) of CD34, a NGFR, or epidermal growth factor receptor(e.g., tEGFR). In some embodiments, the nucleic acid encoding the markeris operably linked to a polynucleotide encoding for a linker sequence,such as a cleavable linker sequence, e.g., T2A. For example, a marker,and optionally a linker sequence, can be any as disclosed in publishedpatent application No. WO2014031687. For example, the marker can be atruncated EGFR (tEGFR) that is, optionally, linked to a linker sequence,such as a T2A cleavable linker sequence. An exemplary polypeptide for atruncated EGFR (e.g. tEGFR) comprises the sequence of amino acids setforth in SEQ ID NO:8 or a sequence of amino acids that exhibits at least85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or more sequence identity to SEQ ID NO: 19.

In some embodiments, a single promoter may direct expression of an RNAthat contains, in a single open reading frame (ORF), two or three genes(e.g. encoding the molecule involved in modulating a metabolic pathwayand encoding the recombinant receptor) separated from one another bysequences encoding a self-cleavage peptide (e.g., 2A sequences) or aprotease recognition site (e.g., furin). The ORF thus encodes a singlepolypeptide, which, either during (in the case of 2A) or aftertranslation, is processed into the individual proteins. In some cases,the peptide, such as T2A, can cause the ribosome to skip (ribosomeskipping) synthesis of a peptide bond at the C-terminus of a 2A element,leading to separation between the end of the 2A sequence and the nextpeptide downstream (see, for example, de Felipe. Genetic Vaccines andTher. 2:13 (2004) and deFelipe et al. Traffic 5:616-626 (2004)). Many 2Aelements are known. Examples of 2A sequences that can be used in themethods and nucleic acids disclosed herein, without limitation, 2Asequences from the foot-and-mouth disease virus (F2A, e.g., SEQ ID NO:24), equine rhinitis A virus (E2A, e.g., SEQ ID NO: 23), Thosea asignavirus (T2A, e.g., SEQ ID NO: 7 or 20), and porcine teschovirus-1 (P2A,e.g., SEQ ID NO: 21 or 22) as described in U.S. Patent Publication No.20070116690. An exemplary T2A linker sequence comprises the sequence ofamino acids set forth in SEQ ID NO: 7 or a sequence of amino acids thatexhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 7.

In some embodiments, the marker is a molecule, e.g., cell surfaceprotein, not naturally found on T cells or not naturally found on thesurface of T cells, or a portion thereof. In some embodiments, themolecule is a non-self molecule, e.g., non-self protein, i.e., one thatis not recognized as “self” by the immune system of the host into whichthe cells will be adoptively transferred.

In some embodiments, the marker is a transduction marker or a surrogatemarker. A transduction marker or a surrogate marker can be used todetect cells that have been introduced with the polynucleotide, e.g., apolynucleotide encoding a recombinant receptor. In some embodiments, thetransduction marker can indicate or confirm modification of a cell. Insome embodiments, the surrogate marker is a protein that is made to beco-expressed on the cell surface with the recombinant receptor, e.g.CAR. In particular embodiments, such a surrogate marker is a surfaceprotein that has been modified to have little or no activity. In certainembodiments, the surrogate marker is encoded on the same polynucleotidethat encodes the recombinant receptor. In some embodiments, the nucleicacid sequence encoding the recombinant receptor is operably linked to anucleic acid sequence encoding a marker, optionally separated by aninternal ribosome entry site (IRES), or a nucleic acid encoding aself-cleaving peptide or a peptide that causes ribosome skipping, suchas a 2A sequence, such as a T2A, a P2A, an E2A or an F2A. Extrinsicmarker genes may in some cases be utilized in connection with engineeredcell to permit detection or selection of cells and, in some cases, alsoto promote cell suicide.

Exemplary surrogate markers can include truncated forms of cell surfacepolypeptides, such as truncated forms that are non-functional and to nottransduce or are not capable of transducing a signal or a signalordinarily transduced by the full-length form of the cell surfacepolypeptide, and/or do not or are not capable of internalizing.Exemplary truncated cell surface polypeptides including truncated formsof growth factors or other receptors such as a truncated human epidermalgrowth factor receptor 2 (tHER2), a truncated epidermal growth factorreceptor (tEGFR, exemplary tEGFR sequence set forth in SEQ ID NO: 8 or19) or a prostate-specific membrane antigen (PSMA) or modified formthereof. tEGFR may contain an epitope recognized by the antibodycetuximab (Erbitux®) or other therapeutic anti-EGFR antibody or bindingmolecule, which can be used to identify or select cells that have beenengineered with the tEGFR construct and an encoded exogenous protein,and/or to eliminate or separate cells expressing the encoded exogenousprotein. See U.S. Pat. No. 8,802,374 and Liu et al., Nature Biotech.2016 April; 34(4): 430-434). In some aspects, the marker, e.g. surrogatemarker, includes all or part (e.g., truncated form) of CD34, a NGFR, aCD19 or a truncated CD19, e.g., a truncated non-human CD19, or epidermalgrowth factor receptor (e.g., tEGFR). In some embodiments, the marker isor comprises a fluorescent protein, such as green fluorescent protein(GFP), enhanced green fluorescent protein (EGFP), such as super-fold GFP(sfGFP), red fluorescent protein (RFP), such as tdTomato, mCherry,mStrawberry, AsRed2, DsRed or DsRed2, cyan fluorescent protein (CFP),blue green fluorescent protein (BFP), enhanced blue fluorescent protein(EBFP), and yellow fluorescent protein (YFP), and variants thereof,including species variants, monomeric variants, and codon-optimizedand/or enhanced variants of the fluorescent proteins. In someembodiments, the marker is or comprises an enzyme, such as a luciferase,the lacZ gene from E. coli, alkaline phosphatase, secreted embryonicalkaline phosphatase (SEAP), chloramphenicol acetyl transferase (CAT).Exemplary light-emitting reporter genes include luciferase (luc),β-galactosidase, chloramphenicol acetyltransferase (CAT),β-glucuronidase (GUS) or variants thereof.

In some embodiments, the marker is a selection marker. In someembodiments, the selection marker is or comprises a polypeptide thatconfers resistance to exogenous agents or drugs. In some embodiments,the selection marker is an antibiotic resistance gene. In someembodiments, the selection marker is an antibiotic resistance geneconfers antibiotic resistance to a mammalian cell. In some embodiments,the selection marker is or comprises a Puromycin resistance gene, aHygromycin resistance gene, a Blasticidin resistance gene, a Neomycinresistance gene, a Geneticin resistance gene or a Zeocin resistance geneor a modified form thereof.

In some embodiments, the nucleic acid encoding the marker is operablylinked to a polynucleotide encoding for a linker sequence, such as acleavable linker sequence, e.g., a T2A. For example, a marker, andoptionally a linker sequence, can be any as disclosed in PCT Pub. No.WO2014031687. For example, the marker can be a truncated EGFR (tEGFR)that is, optionally, linked to a linker sequence, such as a T2Acleavable linker sequence. An exemplary polypeptide for a truncated EGFR(e.g. tEGFR) comprises the sequence of amino acids set forth in SEQ IDNO: 8 or 19 or a sequence of amino acids that exhibits at least 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% ormore sequence identity to SEQ ID NO: 8 or 19.

In some embodiments, the marker serves no therapeutic function and/orproduces no effect other than to be used as a marker for geneticengineering, e.g., for selecting cells successfully engineered. In otherembodiments, the marker may be a therapeutic molecule or moleculeotherwise exerting some desired effect, such as a ligand for a cell tobe encountered in vivo, such as a costimulatory or immune checkpointmolecule to enhance and/or dampen responses of the cells upon adoptivetransfer and encounter with ligand.

In some cases, CARs are referred to as first, second, and/or thirdgeneration CARs. In some aspects, a first generation CAR is one thatsolely provides a CD3-chain induced signal upon antigen binding; in someaspects, a second-generation CARs is one that provides such a signal andcostimulatory signal, such as one including an intracellular signalingdomain from a costimulatory receptor such as CD28 or CD137; in someaspects, a third generation CAR is one that includes multiplecostimulatory domains of different costimulatory receptors.

In some embodiments, the chimeric antigen receptor includes anextracellular portion containing an antibody or antibody fragment. Insome aspects, the chimeric antigen receptor includes an extracellularportion containing the antibody or fragment and an intracellularsignaling domain. In some embodiments, the antibody or fragment includesan scFv and the intracellular domain contains an ITAM. In some aspects,the intracellular signaling domain includes a signaling domain of a zetachain of a CD3-zeta (CD3) chain. In some embodiments, the chimericantigen receptor includes a transmembrane domain linking theextracellular domain and the intracellular signaling domain. In someaspects, the transmembrane domain contains a transmembrane portion ofCD28 or a variant thereof. In some embodiments, the chimeric antigenreceptor contains an intracellular domain of a T cell costimulatorymolecule. The extracellular domain and transmembrane domain can belinked directly or indirectly. In some embodiments, the extracellulardomain and transmembrane are linked by a spacer, such as any describedherein. In some embodiments, the receptor contains extracellular portionof the molecule from which the transmembrane domain is derived, such asa CD28 extracellular portion. In some embodiments, the chimeric antigenreceptor contains an intracellular domain derived from a T cellcostimulatory molecule or a functional variant thereof, such as betweenthe transmembrane domain and intracellular signaling domain. In someaspects, the T cell costimulatory molecule is CD28 or 41BB.

For example, in some embodiments, the CAR contains an antibody, e.g., anantibody fragment, a transmembrane domain that is or contains atransmembrane portion of CD28 or a functional variant thereof, and anintracellular signaling domain containing a signaling portion of CD28 orfunctional variant thereof and a signaling portion of CD3 zeta orfunctional variant thereof. In some embodiments, the CAR contains anantibody, e.g., antibody fragment, a transmembrane domain that is orcontains a transmembrane portion of CD28 or a functional variantthereof, and an intracellular signaling domain containing a signalingportion of a 4-1BB or functional variant thereof and a signaling portionof CD3 zeta or functional variant thereof. In some such embodiments, thereceptor further includes a spacer containing a portion of an Igmolecule, such as a human Ig molecule, such as an Ig hinge, e.g. an IgG4hinge, such as a hinge-only spacer.

In some embodiments, the transmembrane domain of the recombinantreceptor, e.g., the CAR, is or includes a transmembrane domain of humanCD28 (e.g. Accession No. P01747.1) or variant thereof, such as atransmembrane domain that comprises the sequence of amino acids setforth in SEQ ID NO: 9 or a sequence of amino acids that exhibits atleast 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99% or more sequence identity to SEQ ID NO: 9; in some embodiments,the transmembrane-domain containing portion of the recombinant receptorcomprises the sequence of amino acids set forth in SEQ ID NO: 10 or asequence of amino acids having at least at or about 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequenceidentity thereto.

In some embodiments, the intracellular signaling component(s) of therecombinant receptor, e.g. the CAR, contains an intracellularcostimulatory signaling domain of human CD28 or a functional variant orportion thereof, such as a domain with an LL to GG substitution atpositions 186-187 of a native CD28 protein. For example, theintracellular signaling domain can comprise the sequence of amino acidsset forth in SEQ ID NO: 11 or 12 or a sequence of amino acids thatexhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 11 or 12. Insome embodiments, the intracellular domain comprises an intracellularcostimulatory signaling domain of 4-1BB (e.g. (Accession No. Q07011.1)or functional variant or portion thereof, such as the sequence of aminoacids set forth in SEQ ID NO: 13 or a sequence of amino acids thatexhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 13.

In some embodiments, the intracellular signaling domain of therecombinant receptor, e.g. the CAR, comprises a human CD3 zetastimulatory signaling domain or functional variant thereof, such as an112 AA cytoplasmic domain of isoform 3 of human CD3 (Accession No.:P20963.2) or a CD3 zeta signaling domain as described in U.S. Pat. Nos.7,446,190 or 8,911,993. For example, in some embodiments, theintracellular signaling domain comprises the sequence of amino acids asset forth in SEQ ID NO: 14, 15, or 16 or a sequence of amino acids thatexhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 14, 15, or16.

In some aspects, the spacer contains only a hinge region of an IgG, suchas only a hinge of IgG4 or IgG1, such as the hinge only spacer set forthin SEQ ID NO: 2. In other embodiments, the spacer is or contains an Ighinge, e.g., an IgG4-derived hinge, optionally linked to a CH2 and/orCH3 domains. In some embodiments, the spacer is an Ig hinge, e.g., anIgG4 hinge, linked to CH2 and CH3 domains, such as set forth in SEQ IDNO: 5. In some embodiments, the spacer is an Ig hinge, e.g., an IgG4hinge, linked to a CH3 domain only, such as set forth in SEQ ID NO: 4.In some embodiments, the spacer is or comprises a glycine-serine richsequence or other flexible linker such as known flexible linkers.

For example, in some embodiments, the CAR includes an antibody such asan antibody fragment, including scFvs, a spacer, such as a spacercontaining a portion of an immunoglobulin molecule, such as a hingeregion and/or one or more constant regions of a heavy chain molecule,such as an Ig-hinge containing spacer, a transmembrane domain containingall or a portion of a CD28-derived transmembrane domain, a CD28-derivedintracellular signaling domain, and a CD3 zeta signaling domain. In someembodiments, the CAR includes an antibody or fragment, such as scFv, aspacer such as any of the Ig-hinge containing spacers, a CD28-derivedtransmembrane domain, a 4-1BB-derived intracellular signaling domain,and a CD3 zeta-derived signaling domain.

In some embodiments, nucleic acid molecules encoding such CAR constructsfurther includes a sequence encoding a T2A ribosomal skip element and/ora tEGFR sequence, e.g., downstream of the sequence encoding the CAR. Insome embodiments, the sequence encodes a T2A ribosomal skip element setforth in SEQ ID NO: 7, or a sequence of amino acids that exhibits atleast 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99% or more sequence identity to SEQ ID NO: 7. In some embodiments,T cells expressing an antigen receptor (e.g. CAR) can also be generatedto express a truncated EGFR (EGFRt) as a non-immunogenic selectionepitope (e.g. by introduction of a construct encoding the CAR and EGFRtseparated by a T2A ribosome switch to express two proteins from the sameconstruct), which then can be used as a marker to detect such cells (seee.g. U.S. Pat. No. 8,802,374). In some embodiments, the sequence encodesan tEGFR sequence set forth in SEQ ID NO: 8, or a sequence of aminoacids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ IDNO: 8.

The recombinant receptors, such as CARs, expressed by the cellsadministered to the subject generally recognize or specifically bind toa molecule that is expressed in, associated with, and/or specific forthe disease or condition or cells thereof being treated. Upon specificbinding to the molecule, e.g., antigen, the receptor generally deliversan immunostimulatory signal, such as an ITAM-transduced signal, into thecell, thereby promoting an immune response targeted to the disease orcondition. For example, in some embodiments, the cells express a CARthat specifically binds to an antigen expressed by a cell or tissue ofthe disease or condition or associated with the disease or condition.

2. TCRs

In certain embodiments, the surface expression of glycans, e.g.,N-glycans, of cell compositions containing engineered cells that expressa recombinant receptor are assessed by the methods provided herein. Insome embodiments, the expression of surface glycans is assessed inengineered cells, such as T cells, that express a T cell receptor (TCR)or an antigen-binding portion thereof.

In certain embodiments, the methods provided herein may be used toassess the surface glycan profile, i.e., the expression of the surfaceglycans, of a composition of cells that express a recombinant T cellreceptor. In some embodiments, a “T cell receptor” or “TCR” is amolecule that contains a variable α and β chains (also known as TCRα andTCRβ, respectively) or a variable γ and δ chains (also known as TCRα andTCRβ, respectively), or antigen-binding portions thereof, and which iscapable of specifically binding to a peptide bound to an MHC molecule.In some embodiments, the TCR is in the αβ form. Typically, TCRs thatexist in αβ and γδ forms are generally structurally similar, but T cellsexpressing them may have distinct anatomical locations or functions. ATCR can be found on the surface of a cell or in soluble form. Generally,a TCR is found on the surface of T cells (or T lymphocytes) where it isgenerally responsible for recognizing antigens bound to majorhistocompatibility complex (MHC) molecules.

Unless otherwise stated, the term “TCR” should be understood toencompass full TCRs as well as antigen-binding portions orantigen-binding fragments thereof. In some embodiments, the TCR is anintact or full-length TCR, including TCRs in the αβ form or γδ form. Insome embodiments, the TCR is an antigen-binding portion that is lessthan a full-length TCR but that binds to a specific peptide bound in anMHC molecule, such as binds to an MHC-peptide complex. In some cases, anantigen-binding portion or fragment of a TCR can contain only a portionof the structural domains of a full-length or intact TCR, but yet isable to bind the peptide epitope, such as MHC-peptide complex, to whichthe full TCR binds. In some cases, an antigen-binding portion containsthe variable domains of a TCR, such as variable α chain and variable βchain of a TCR, sufficient to form a binding site for binding to aspecific MHC-peptide complex. Generally, the variable chains of a TCRcontain complementarity determining regions involved in recognition ofthe peptide, MHC and/or MHC-peptide complex.

In some embodiments, the variable domains of the TCR containhypervariable loops, or complementarity determining regions (CDRs),which generally are the primary contributors to antigen recognition andbinding capabilities and specificity. In some embodiments, a CDR of aTCR or combination thereof forms all or substantially all of theantigen-binding site of a given TCR molecule. The various CDRs within avariable region of a TCR chain generally are separated by frameworkregions (FRs), which generally display less variability among TCRmolecules as compared to the CDRs (see, e.g., Jores et al., Proc. Nat'lAcad. Sci. U.S.A. 87:9138, 1990; Chothia et al., EMBO J. 7:3745, 1988;see also Lefranc et al., Dev. Comp. Immunol. 27:55, 2003). In someembodiments, CDR3 is the main CDR responsible for antigen binding orspecificity, or is the most important among the three CDRs on a givenTCR variable region for antigen recognition, and/or for interaction withthe processed peptide portion of the peptide-MHC complex. In somecontexts, the CDR1 of the alpha chain can interact with the N-terminalpart of certain antigenic peptides. In some contexts, CDR1 of the betachain can interact with the C-terminal part of the peptide. In somecontexts, CDR2 contributes most strongly to or is the primary CDRresponsible for the interaction with or recognition of the MHC portionof the MHC-peptide complex. In some embodiments, the variable region ofthe β-chain can contain a further hypervariable region (CDR4 or HVR4),which generally is involved in superantigen binding and not antigenrecognition (Kotb (1995) Clinical Microbiology Reviews, 8:411-426).

In some embodiments, a TCR also can contain a constant domain, atransmembrane domain and/or a short cytoplasmic tail (see, e.g., Janewayet al., Immunobiology: The Immune System in Health and Disease, 3rd Ed.,Current Biology Publications, p. 4:33, 1997). In some aspects, eachchain of the TCR can possess one N-terminal immunoglobulin variabledomain, one immunoglobulin constant domain, a transmembrane region, anda short cytoplasmic tail at the C-terminal end. In some embodiments, aTCR is associated with invariant proteins of the CD3 complex involved inmediating signal transduction.

In some embodiments, a TCR chain contains one or more constant domain.For example, the extracellular portion of a given TCR chain (e.g.,α-chain or β-chain) can contain two immunoglobulin-like domains, such asa variable domain (e.g., Vα or Vβ; typically amino acids 1 to 116 basedon Kabat numbering Kabat et al., “Sequences of Proteins of ImmunologicalInterest, US Dept. Health and Human Services, Public Health ServiceNational Institutes of Health, 1991, 5th ed.) and a constant domain(e.g., α-chain constant domain or Cα, typically positions 117 to 259 ofthe chain based on Kabat numbering or β chain constant domain or Cβ,typically positions 117 to 295 of the chain based on Kabat) adjacent tothe cell membrane. For example, in some cases, the extracellular portionof the TCR formed by the two chains contains two membrane-proximalconstant domains, and two membrane-distal variable domains, whichvariable domains each contain CDRs. The constant domain of the TCR maycontain short connecting sequences in which a cysteine residue forms adisulfide bond, thereby linking the two chains of the TCR. In someembodiments, a TCR may have an additional cysteine residue in each ofthe α and β chains, such that the TCR contains two disulfide bonds inthe constant domains.

In some embodiments, the TCR chains contain a transmembrane domain. Insome embodiments, the transmembrane domain is positively charged. Insome cases, the TCR chain contains a cytoplasmic tail. In some cases,the structure allows the TCR to associate with other molecules like CD3and subunits thereof. For example, a TCR containing constant domainswith a transmembrane region may anchor the protein in the cell membraneand associate with invariant subunits of the CD3 signaling apparatus orcomplex. The intracellular tails of CD3 signaling subunits (e.g. CD3γ,CD3δ, CD3ε and CD3ζ chains) contain one or more immunoreceptortyrosine-based activation motif or ITAM that are involved in thesignaling capacity of the TCR complex.

In some embodiments, the TCR may be a heterodimer of two chains α and β(or optionally γ and δ) or it may be a single chain TCR construct. Insome embodiments, the TCR is a heterodimer containing two separatechains (α and β chains or γ and δ chains) that are linked, such as by adisulfide bond or disulfide bonds.

In some embodiments, the TCR can be generated from a known TCRsequence(s), such as sequences of Vα,β chains, for which a substantiallyfull-length coding sequence is readily available. Methods for obtainingfull-length TCR sequences, including V chain sequences, from cellsources are well known. In some embodiments, nucleic acids encoding theTCR can be obtained from a variety of sources, such as by polymerasechain reaction (PCR) amplification of TCR-encoding nucleic acids withinor isolated from a given cell or cells, or synthesis of publiclyavailable TCR DNA sequences.

In some embodiments, the TCR is obtained from a biological source, suchas from cells such as from a T cell (e.g. cytotoxic T cell), T-cellhybridomas or other publicly available source. In some embodiments, theT-cells can be obtained from in vivo isolated cells. In someembodiments, the TCR is a thymically selected TCR. In some embodiments,the TCR is a neoepitope-restricted TCR. In some embodiments, the T-cellscan be a cultured T-cell hybridoma or clone. In some embodiments, theTCR or antigen-binding portion thereof can be synthetically generatedfrom knowledge of the sequence of the TCR.

In some embodiments, the TCR is generated from a TCR identified orselected from screening a library of candidate TCRs against a targetpolypeptide antigen, or target T cell epitope thereof. TCR libraries canbe generated by amplification of the repertoire of Va and VP from Tcells isolated from a subject, including cells present in PBMCs, spleenor other lymphoid organ. In some cases, T cells can be amplified fromtumor-infiltrating lymphocytes (TILs). In some embodiments, TCRlibraries can be generated from CD4+ or CD8+ T cells. In someembodiments, the TCRs can be amplified from a T cell source of a normalof healthy subject, i.e. normal TCR libraries. In some embodiments, theTCRs can be amplified from a T cell source of a diseased subject, i.e.diseased TCR libraries. In some embodiments, degenerate primers are usedto amplify the gene repertoire of Va and VP, such as by RT-PCR insamples, such as T cells, obtained from humans. In some embodiments,scTv libraries can be assembled from naive Va and VP libraries in whichthe amplified products are cloned or assembled to be separated by alinker. Depending on the source of the subject and cells, the librariescan be HLA allele-specific. Alternatively, in some embodiments, TCRlibraries can be generated by mutagenesis or diversification of a parentor scaffold TCR molecule. In some aspects, the TCRs are subjected todirected evolution, such as by mutagenesis, e.g., of the α or β chain.In some aspects, particular residues within CDRs of the TCR are altered.In some embodiments, selected TCRs can be modified by affinitymaturation. In some embodiments, antigen-specific T cells may beselected, such as by screening to assess CTL activity against thepeptide. In some aspects, TCRs, e.g. present on the antigen-specific Tcells, may be selected, such as by binding activity, e.g., particularaffinity or avidity for the antigen.

In some embodiments, the TCR or antigen-binding portion thereof is onethat has been modified or engineered. In some embodiments, directedevolution methods are used to generate TCRs with altered properties,such as with higher affinity for a specific MHC-peptide complex. In someembodiments, directed evolution is achieved by display methodsincluding, but not limited to, yeast display (Holler et al. (2003) NatImmunol, 4, 55-62; Holler et al. (2000) Proc Natl Acad Sci USA, 97,5387-92), phage display (Li et al. (2005) Nat Biotechnol, 23, 349-54),or T cell display (Chervin et al. (2008) J Immunol Methods, 339,175-84). In some embodiments, display approaches involve engineering, ormodifying, a known, parent or reference TCR. For example, in some cases,a wild-type TCR can be used as a template for producing mutagenized TCRsin which in one or more residues of the CDRs are mutated, and mutantswith an desired altered property, such as higher affinity for a desiredtarget antigen, are selected.

In some embodiments, peptides of a target polypeptide for use inproducing or generating a TCR of interest are known or can be readilyidentified by a skilled artisan. In some embodiments, peptides suitablefor use in generating TCRs or antigen-binding portions can be determinedbased on the presence of an HLA-restricted motif in a target polypeptideof interest, such as a target polypeptide described below. In someembodiments, peptides are identified using computer prediction modelsknown to those of skill in the art. In some embodiments, for predictingMHC class I binding sites, such models include, but are not limited to,ProPred1 (Singh and Raghava (2001) Bioinformatics 17(12):1236-1237, andSYFPEITHI (see Schuler et al. (2007) Immunoinformatics Methods inMolecular Biology, 409(1): 75-93 2007). In some embodiments, theMHC-restricted epitope is HLA-A0201, which is expressed in approximately39-46% of all Caucasians and therefore, represents a suitable choice ofMHC antigen for use preparing a TCR or other MHC-peptide bindingmolecule.

HLA-A0201-binding motifs and the cleavage sites for proteasomes andimmune-proteasomes using computer prediction models are known to thoseof skill in the art. For predicting MHC class I binding sites, suchmodels include, but are not limited to, ProPred1 (described in moredetail in Singh and Raghava, ProPred: prediction of HLA-DR bindingsites. BIOINFORMATICS 17(12):1236-1237 2001), and SYFPEITHI (see Schuleret al. SYFPEITHI, Database for Searching and T-Cell Epitope Prediction.in Immunoinformatics Methods in Molecular Biology, vol 409(1): 75-932007)

In some embodiments, the TCR or antigen binding portion thereof may be arecombinantly produced natural protein or mutated form thereof in whichone or more property, such as binding characteristic, has been altered.In some embodiments, a TCR may be derived from one of various animalspecies, such as human, mouse, rat, or other mammal. A TCR may becell-bound or in soluble form. In some embodiments, for purposes of theprovided methods, the TCR is in cell-bound form expressed on the surfaceof a cell.

In some embodiments, the TCR is a full-length TCR. In some embodiments,the TCR is an antigen-binding portion. In some embodiments, the TCR is adimeric TCR (dTCR). In some embodiments, the TCR is a single-chain TCR(sc-TCR). In some embodiments, a dTCR or scTCR have the structures asdescribed in WO 03/020763, WO 04/033685, WO2011/044186.

In some embodiments, the TCR contains a sequence corresponding to thetransmembrane sequence. In some embodiments, the TCR does contain asequence corresponding to cytoplasmic sequences. In some embodiments,the TCR is capable of forming a TCR complex with CD3. In someembodiments, any of the TCRs, including a dTCR or scTCR, can be linkedto signaling domains that yield an active TCR on the surface of a Tcell. In some embodiments, the TCR is expressed on the surface of cells.

In some embodiments a dTCR contains a first polypeptide wherein asequence corresponding to a TCR α chain variable region sequence isfused to the N terminus of a sequence corresponding to a TCR α chainconstant region extracellular sequence, and a second polypeptide whereina sequence corresponding to a TCR β chain variable region sequence isfused to the N terminus a sequence corresponding to a TCR β chainconstant region extracellular sequence, the first and secondpolypeptides being linked by a disulfide bond. In some embodiments, thebond can correspond to the native inter-chain disulfide bond present innative dimeric αβ TCRs. In some embodiments, the interchain disulfidebonds are not present in a native TCR. For example, in some embodiments,one or more cysteines can be incorporated into the constant regionextracellular sequences of dTCR polypeptide pair. In some cases, both anative and a non-native disulfide bond may be desirable. In someembodiments, the TCR contains a transmembrane sequence to anchor to themembrane.

In some embodiments, a dTCR contains a TCR α chain containing a variableα domain, a constant α domain and a first dimerization motif attached tothe C-terminus of the constant α domain, and a TCR β chain comprising avariable β domain, a constant β domain and a first dimerization motifattached to the C-terminus of the constant β domain, wherein the firstand second dimerization motifs easily interact to form a covalent bondbetween an amino acid in the first dimerization motif and an amino acidin the second dimerization motif linking the TCR α chain and TCR β chaintogether.

In some embodiments, the TCR is a scTCR. Typically, a scTCR can begenerated using methods known to those of skill in the art, See e.g.,Soo Hoo, W. F. et al. PNAS (USA) 89, 4759 (1992); Wülfing, C. andPlückthun, A., J. Mol. Biol. 242, 655 (1994); Kurucz, I. et al. PNAS(USA) 90 3830 (1993); International published PCT Nos. WO 96/13593, WO96/18105, WO99/60120, WO99/18129, WO 03/020763, WO2011/044186; andSchlueter, C. J. et al. J. Mol. Biol. 256, 859 (1996). In someembodiments, a scTCR contains an introduced non-native disulfideinterchain bond to facilitate the association of the TCR chains (seee.g. International published PCT No. WO 03/020763). In some embodiments,a scTCR is a non-disulfide linked truncated TCR in which heterologousleucine zippers fused to the C-termini thereof facilitate chainassociation (see e.g. International published PCT No. WO99/60120). Insome embodiments, a scTCR contain a TCRa variable domain covalentlylinked to a TCRβ variable domain via a peptide linker (see e.g.,International published PCT No. WO99/18129).

In some embodiments, a scTCR contains a first segment constituted by anamino acid sequence corresponding to a TCR α chain variable region, asecond segment constituted by an amino acid sequence corresponding to aTCR β chain variable region sequence fused to the N terminus of an aminoacid sequence corresponding to a TCR β chain constant domainextracellular sequence, and a linker sequence linking the C terminus ofthe first segment to the N terminus of the second segment.

In some embodiments, a scTCR contains a first segment constituted by ana chain variable region sequence fused to the N terminus of an a chainextracellular constant domain sequence, and a second segment constitutedby a β chain variable region sequence fused to the N terminus of asequence β chain extracellular constant and transmembrane sequence, and,optionally, a linker sequence linking the C terminus of the firstsegment to the N terminus of the second segment.

In some embodiments, a scTCR contains a first segment constituted by aTCR β chain variable region sequence fused to the N terminus of a βchain extracellular constant domain sequence, and a second segmentconstituted by an a chain variable region sequence fused to the Nterminus of a sequence α chain extracellular constant and transmembranesequence, and, optionally, a linker sequence linking the C terminus ofthe first segment to the N terminus of the second segment.

In some embodiments, the linker of a scTCRs that links the first andsecond TCR segments can be any linker capable of forming a singlepolypeptide strand, while retaining TCR binding specificity. In someembodiments, the linker sequence may, for example, have the formula-P-AA-P- wherein P is proline and AA represents an amino acid sequencewherein the amino acids are glycine and serine. In some embodiments, thefirst and second segments are paired so that the variable regionsequences thereof are orientated for such binding. Hence, in some cases,the linker has a sufficient length to span the distance between the Cterminus of the first segment and the N terminus of the second segment,or vice versa, but is not too long to block or reduces bonding of thescTCR to the target ligand. In some embodiments, the linker can containfrom or from about 10 to 45 amino acids, such as 10 to 30 amino acids or26 to 41 amino acids residues, for example 29, 30, 31 or 32 amino acids.In some embodiments, the linker has the formula -PGGG-(SGGGG)5-P-wherein P is proline, G is glycine and S is serine (SEQ ID NO: 17). Insome embodiments, the linker has the sequence GSADDAKKDAAKKDGKS (SEQ IDNO: 18).

In some embodiments, the scTCR contains a covalent disulfide bondlinking a residue of the immunoglobulin region of the constant domain ofthe α chain to a residue of the immunoglobulin region of the constantdomain of the β chain. In some embodiments, the interchain disulfidebond in a native TCR is not present. For example, in some embodiments,one or more cysteines can be incorporated into the constant regionextracellular sequences of the first and second segments of the scTCRpolypeptide. In some cases, both a native and a non-native disulfidebond may be desirable.

In some embodiments of a dTCR or scTCR containing introduced interchaindisulfide bonds, the native disulfide bonds are not present. In someembodiments, the one or more of the native cysteines forming a nativeinterchain disulfide bonds are substituted to another residue, such asto a serine or alanine. In some embodiments, an introduced disulfidebond can be formed by mutating non-cysteine residues on the first andsecond segments to cysteine. Exemplary non-native disulfide bonds of aTCR are described in published International PCT No. WO2006/000830.

In some embodiments, the TCR or antigen-binding fragment thereofexhibits an affinity with an equilibrium binding constant for a targetantigen of between or between about 10-5 and 10-12 M and all individualvalues and ranges therein. In some embodiments, the target antigen is anMHC-peptide complex or ligand.

In some embodiments, nucleic acid or nucleic acids encoding a TCR, suchas α and β chains, can be amplified by PCR, cloning or other suitablemeans and cloned into a suitable expression vector or vectors. Theexpression vector can be any suitable recombinant expression vector, andcan be used to transform or transfect any suitable host. Suitablevectors include those designed for propagation and expansion or forexpression or both, such as plasmids and viruses.

In some embodiments, the vector can a vector of the pUC series(Fermentas Life Sciences), the pBluescript series (Stratagene, LaJolla,Calif.), the pET series (Novagen, Madison, Wis.), the pGEX series(Pharmacia Biotech, Uppsala, Sweden), or the pEX series (Clontech, PaloAlto, Calif.). In some cases, bacteriophage vectors, such as λG10,λGT11, λZapII (Stratagene), λEMBL4, and λNM1149, also can be used. Insome embodiments, plant expression vectors can be used and includepBI01, pBI101.2, pBI101.3, pBI121 and pBIN19 (Clontech). In someembodiments, animal expression vectors include pEUK-Cl, pMAM and pMAMneo(Clontech). In some embodiments, a viral vector is used, such as aretroviral vector.

In some embodiments, the recombinant expression vectors can be preparedusing standard recombinant DNA techniques. In some embodiments, vectorscan contain regulatory sequences, such as transcription and translationinitiation and termination codons, which are specific to the type ofhost (e.g., bacterium, fungus, plant, or animal) into which the vectoris to be introduced, as appropriate and taking into considerationwhether the vector is DNA- or RNA-based. In some embodiments, the vectorcan contain a nonnative promoter operably linked to the nucleotidesequence encoding the TCR or antigen-binding portion (or otherMHC-peptide binding molecule). In some embodiments, the promoter can bea non-viral promoter or a viral promoter, such as a cytomegalovirus(CMV) promoter, an SV40 promoter, an RSV promoter, and a promoter foundin the long-terminal repeat of the murine stem cell virus. Otherpromoters known to a skilled artisan also are contemplated.

In some embodiments, to generate a vector encoding a TCR, the α and βchains are PCR amplified from total cDNA isolated from a T cell cloneexpressing the TCR of interest and cloned into an expression vector. Insome embodiments, the α and β chains are cloned into the same vector. Insome embodiments, the α and β chains are cloned into different vectors.In some embodiments, the generated α and β chains are incorporated intoa retroviral, e.g. lentiviral, vector.

3. Multi-Targeting

In some embodiments, the surface glycan expression is assessed in cellcompositions that contain cells designed for multi-targeting strategies.In some embodiments, the cell compositions contain cells designed formulti-targeting strategies such as cells that express two or moregenetically engineered receptors on the cell, with each receptorrecognizing the same or a different antigen and typically each includinga different intracellular signaling component. Such multi-targetingstrategies are described, for example, in International PatentApplication, Publication No.: WO 2014055668 A1 (describing combinationsof activating and costimulatory CARs, e.g., targeting two differentantigens present individually on off-target, e.g., normal cells, butpresent together only on cells of the disease or condition to betreated) and Fedorov et al., Sci. Transl. Medicine, 5(215) (2013)(describing cells expressing an activating and an inhibitory CAR, suchas those in which the activating CAR binds to one antigen expressed onboth normal or non-diseased cells and cells of the disease or conditionto be treated, and the inhibitory CAR binds to another antigen expressedonly on the normal cells or cells which it is not desired to treat).

For example, in some embodiments, surface glycans are released andanalyzed from a cell composition that contains the cells that include areceptor expressing a first genetically engineered antigen receptor(e.g., CAR or TCR) which is capable of inducing an activating signal tothe cell, generally upon specific binding to the antigen recognized bythe first receptor, e.g., the first antigen. In some embodiments, thecell further includes a second genetically engineered antigen receptor(e.g., CAR or TCR), e.g., a chimeric costimulatory receptor, which iscapable of inducing a costimulatory signal to the immune cell, generallyupon specific binding to a second antigen recognized by the secondreceptor. In some embodiments, the first antigen and second antigen arethe same. In some embodiments, the first antigen and second antigen aredifferent.

In some embodiments, the first and/or second genetically engineeredantigen receptor (e.g. CAR or TCR) is capable of inducing an activatingsignal to the cell. In some embodiments, the receptor includes anintracellular signaling component containing ITAM or ITAM-like motifs.In some embodiments, the activation induced by the first receptorinvolves a signal transduction or change in protein expression in thecell resulting in initiation of an immune response, such as ITAMphosphorylation and/or initiation of ITAM-mediated signal transductioncascade, formation of an immunological synapse and/or clustering ofmolecules near the bound receptor (e.g. CD4 or CD8, etc.), activation ofone or more transcription factors, such as NF—KB and/or AP-1, and/orinduction of gene expression of factors such as cytokines,proliferation, and/or survival.

In some embodiments, the first and/or second receptor includesintracellular signaling domains of costimulatory receptors such as CD28,CD137 (4-1 BB), OX40, and/or ICOS. In some embodiments, the first andsecond receptor include an intracellular signaling domain of acostimulatory receptor that are different. In one embodiment, the firstreceptor contains a CD28 costimulatory signaling region and the secondreceptor contain a 4-1BB co-stimulatory signaling region or vice versa.

In some embodiments, the first and/or second receptor includes both anintracellular signaling domain containing ITAM or ITAM-like motifs andan intracellular signaling domain of a costimulatory receptor.

In some embodiments, the first receptor contains an intracellularsignaling domain containing ITAM or ITAM-like motifs and the secondreceptor contains an intracellular signaling domain of a costimulatoryreceptor. The costimulatory signal in combination with the activatingsignal induced in the same cell is one that results in an immuneresponse, such as a robust and sustained immune response, such asincreased gene expression, secretion of cytokines and other factors, andT cell mediated effector functions such as cell killing.

In some embodiments, neither ligation of the first receptor alone norligation of the second receptor alone induces a robust immune response.In some aspects, if only one receptor is ligated, the cell becomestolerized or unresponsive to antigen, or inhibited, and/or is notinduced to proliferate or secrete factors or carry out effectorfunctions. In some such embodiments, however, when the plurality ofreceptors are ligated, such as upon encounter of a cell expressing thefirst and second antigens, a desired response is achieved, such as fullimmune activation or stimulation, e.g., as indicated by secretion of oneor more cytokine, proliferation, persistence, and/or carrying out animmune effector function such as cytotoxic killing of a target cell.

In some embodiments, the two receptors induce, respectively, anactivating and an inhibitory signal to the cell, such that binding byone of the receptor to its antigen activates the cell or induces aresponse, but binding by the second inhibitory receptor to its antigeninduces a signal that suppresses or dampens that response. Examples arecombinations of activating CARs and inhibitory CARs or iCARs. Such astrategy may be used, for example, in which the activating CAR binds anantigen expressed in a disease or condition but which is also expressedon normal cells, and the inhibitory receptor binds to a separate antigenwhich is expressed on the normal cells but not cells of the disease orcondition.

In some embodiments, the multi-targeting strategy is employed in a casewhere an antigen associated with a particular disease or condition isexpressed on a non-diseased cell and/or is expressed on the engineeredcell itself, either transiently (e.g., upon stimulation in associationwith genetic engineering) or permanently. In such cases, by requiringligation of two separate and individually specific antigen receptors,specificity, selectivity, and/or efficacy may be improved.

In some embodiments, the plurality of antigens, e.g., the first andsecond antigens, are expressed on the cell, tissue, or disease orcondition being targeted, such as on the cancer cell. In some aspects,the cell, tissue, disease or condition is multiple myeloma or a multiplemyeloma cell. In some embodiments, one or more of the plurality ofantigens generally also is expressed on a cell which it is not desiredto target with the cell therapy, such as a normal or non-diseased cellor tissue, and/or the engineered cells themselves. In such embodiments,by requiring ligation of multiple receptors to achieve a response of thecell, specificity and/or efficacy are achieved.

III. PROCESS FOR MANUFACTURING OR PREPARING CELL COMPOSITIONS

In particular embodiments, the methods provided herein can be used toassess the surface expression of glycans, e.g., N-glycans, of one ormore compositions at various stages of a process for manufacturing orgenerating engineered cells. In particular embodiments, the process maybe for the manufacturing or generation of engineered cells that expressa recombinant receptor, e.g., a CAR or TCR, as described herein such asin Section III. In some embodiments, the process includes steps forisolation, separation, selection, cultivation (e.g., stimulation of thecells, for example, to induce their proliferation and/or activation),transduction and/or transfection, washing, suspension, dilution,concentration, and/or formulation of the cells. In certain embodiments,surface glycan expression is assessed in one or more cell compositionsthat are obtained prior to, in connection with, or after, steps forisolation, separation, selection, cultivation (e.g., stimulation of thecells, for example, to induce their proliferation and/or activation),transduction and/or transfection, washing, suspension, dilution,concentration, and/or formulation of the cells. In particularembodiments, cells may be collected at any stage or between any stagesin the process of manufacturing or generating engineered cells. In someembodiments, the cells may be stored, e.g., cryopreserved, for a lateranalysis of surface glycans, or may be directly incubated underconditions to release surface glycans.

In some embodiments, glycans, e.g., N-glycans, that are released fromthe surface of cells from cell compositions taken at various steps inthe process for generating or manufacturing engineered cells areanalyzed. In some embodiments, the compositions include a cellcomposition that contains PBMCs. In certain embodiments, thecompositions include a cell composition contains selected CD4+ or CD8+ Tcells. In some embodiments, the cell compositions include a cellcomposition that contains activated T cell compositions. In certainembodiments, the T cell composition contains cells that were activatedand transduced or transfected with a vector encoding a recombinantreceptor. In particular embodiments, the cell composition containsexpanded recombinant receptor-expressing cells.

In certain embodiments, the surface expression of glycans, e.g.,N-glycans are assessed from cells of cell compositions taken at varioussteps in the process for generating or manufacturing engineered cells.In some embodiments, the cell compositions include compositions thatcontain bulk PBMCs. In certain embodiments, the cell compositionsinclude a composition that contains thawed cryopreserved cellcompositions containing selected CD4+ or CD8+ T cells selected byimmunoaffinity-based selection. In some embodiments, the compositionsinclude a composition that contains T cells incubated withanti-CD3/anti-CD28 beads in the presence of IL-2, IL-15 and/or IL-7 for18-24 hours at 37° C. In certain embodiments, the cell compositionsinclude a composition that contains activated T cells that weretransduced with a lentiviral vector encoding a CAR. In certainembodiments, the cell compositions include a composition that containsthawed, cryopreserved cell compositions containing CAR-expressing cellsthat were subjected to expansion in the presence of IL-2, IL-15 and/orIL-17 cytokines prior to cryopreservation. In some embodiments, the cellcomposition is a thawed cryopreserved drug product.

In particular embodiments, the methods provided herein may be used toassess the surface expression of glycans, e.g., N-glycans, of cells fromcompositions collected during a process for generating or manufacturingengineered cells. In some embodiments, the cell compositions arecomposed of cells at a stage prior to preforming a step for isolation,separation, selection, cultivation, activation, transduction and/ortransfection, expansion, washing, suspension, dilution, concentration,and/or formulation of the cells. In certain embodiments, the cellcompositions are composed of cells following a step for isolation,separation, selection, cultivation, activation, transduction and/ortransfection, expansion, washing, suspension, dilution, concentration,and/or formulation of the cells. In certain embodiments, the cellcompositions are composed of cells undergoing a step for isolation,separation, selection, cultivation, activation, transduction and/ortransfection, expansion, washing, suspension, dilution, concentration,and/or formulation of the cells.

In some embodiments, the methods provided herein may be used to assessthe surface expression of glycans, e.g., N-glycans, of cells fromcompositions collected during a process for generating or manufacturingengineered cells, for example to compare the glycan surface expressionwith glycan surface expression of a different cell composition. In someembodiments, the surface glycan composition is obtained from acomposition of cells at or undergoing a step in the process, and theexpression of the glycans is compared to the surface glycan expressionof a cell composition at or undergoing a prior step in the process. Incertain embodiments, the surface glycan expression is obtained from acomposition of cells at or undergoing a step in the process, and thesurface glycan expression is compared to the surface glycan expressionof a composition of cells that is at or undergoing a step that isdownstream in the process. In some embodiments, the surface glycanexpression is obtained from a composition of cells at or undergoing astep in the process, and the surface glycan expression is compared tothe surface glycan expression of a composition of cells has completedthe process. In particular embodiments, the surface glycan expression isobtained from a composition of cells at or undergoing a step in theprocess, and the surface glycan expression is compared to the surfaceglycan expression of a composition of cells has not undergone theprocess.

In particular embodiments, the methods provided herein may be used toassess the surface expression of glycans, e.g., N-glycans, of cells fromcompositions that have completed a process for generating ormanufacturing engineered cells. In some embodiments, compositions ofcells have completed the same process and express the same one or morerecombinant receptors. In certain embodiments, the compositions of cellshave completed the same process and express different one or morerecombinant receptors. In some embodiments, surface glycan expression iscompared between two or more cell compositions that have completed thesame process and express the same one or more recombinant receptors.

Particular embodiments contemplate that it may be desirable fordifferent cell compositions that express the same recombinant receptorand that have undergone the same process to have similar surface glycanexpression. In some embodiments, surface glycan expressions of aplurality of cell compositions that express the same recombinantreceptor and that have undergone the same process are measured and/orassessed. In some embodiments, the methods can be employed in a releaseassay to confirm consistency and/or substantial homogeneity of a cellcomposition prior to release of a cell composition, e.g. therapeuticcomposition, for administration to a subject. In some embodiments, thesurface glycan profile, e.g. presence, absence, identity and/or level ofone or more glycans, in a sample from a cell composition are compared toa reference standard that is or indicates a release specification, alabel requirement and/or a compendia specification.

In some embodiments, a reference standard comprises an average or medianof the presence, absence, identity and/or level of the one or moretarget glycan or glycans among a plurality of compositions produced bythe process. In particular embodiments, the mean, median, and/orvariance of a measurement of expression of one or more target glycan orglycans, e.g. glycan species, families, and/or groups, are calculatedfor a plurality of the cell compositions. In some embodiments, thepresence, absence, identity and/or level of one or more target glycan orglycans in a sample released from a cell composition, e.g. test cellcomposition, according to the provided methods is compared to suchaverage or median of the target glycan or glycans.

In some embodiments, the cell composition is released for treatment of asubject only if the cell surface glycan profile of the composition issubstantially the same as the reference sample and/or if the percent ofa target glycan or each of a plurality of target glycans to the totalglycans present in the sample differs by no more than 25%, no more than20% or no more than 10% from the percent of the target glycan or each ofthe plurality of target glycans to the total glycans present in thereference sample.

In some embodiments, one or more parameters of one or more steps of theprocess for manufacturing or generating the engineered cells areadjusted, altered, and/or changed if the variance of the expression ofone or more glycan species, families, and/or groups is greater than athreshold. In some embodiments, the threshold is a variation of ±50%,±33%, ±25%, ±20%, ±15%, ±10%, ±7.5%, ±5%, ±4%, ±3%, ±2%, ±1%, ±0.1%,±0.05%, or ±0.01% of a measurement of the expression of one or moreglycan species, families, and/or groups. In certain embodiments, themeasurement of the expression is the relative amount of the one or moreglycan species, families, and/or groups, i.e., the percentage of theexpression of the one or more glycan species, families, and/or groups ofthe total surface glycan expression. Examples of parameters that may bealtered to improve consistency and/or reduce variation of surface glycanexpression includes timing and/or duration of incubations,concentrations of cell preparations, additional or removal of reagents,and concentrations of reagents.

In some embodiments, the provided methods are carried out on one or morecompositions produced in connection with the preparation ormanufacturing of a cell composition containing engineered cells. In someembodiments, the manufacturing process comprises one or more stages orsteps that result in cells or a cell composition containing cellsisolated from a biological sample by leukapheresis or apheresis; cellsselected from a biological sample by immunoaffinity-based methods; cellsintroduced with a recombinant nucleic acid, optionally a viral vectorencoding a recombinant protein; cell incubated in the presence of one ormore test agents, optionally one more peptide, protein, polypeptide,nucleic acid, small molecule; cells activated, expanded in the presenceof one or more stimulating conditions; and/or cryopreservation of cellsin the presence of a cryoprotectant; and/or cells formulated foradministration to a subject, optionally in the presence of apharmaceutically acceptable excipient.

1. Preparation of Cells for Genetic Engineering

Among the cells that may be assessed for glycan surface expression bythe methods provided herein are engineered cells that expressrecombinant receptors. In certain embodiments, the surface glycanexpression may be assessed by the methods provided herein from one ormore compositions of cells collected from various stages of the geneticengineering process.

The genetic engineering generally involves introduction of a nucleicacid encoding the recombinant or engineered component into a compositioncontaining the cells, such as by retroviral transduction, transfection,or transformation. In some embodiments, surface glycans are assessed incompositions of cells that are collected prior the start of theretroviral transduction, transfection, or transformation. In certainembodiments, surface glycans are assessed in compositions of cells thatare collected immediately after the process of the retroviraltransduction, transfection, or transformation. In particularembodiments, the surface glycans are assessed in compositions of cellsthat are collected at one or more stages of the retroviral transduction,transfection, or transformation.

In some embodiments, the nucleic acids are heterologous, i.e., normallynot present in a cell or sample obtained from the cell, such as oneobtained from another organism or cell, which for example, is notordinarily found in the cell being engineered and/or an organism fromwhich such cell is derived. In some embodiments, the nucleic acids arenot naturally occurring, such as a nucleic acid not found in nature,including one comprising chimeric combinations of nucleic acids encodingvarious domains from multiple different cell types.

The cells generally are eukaryotic cells, such as mammalian cells, andtypically are human cells. In some embodiments, the cells are derivedfrom the blood, bone marrow, lymph, or lymphoid organs, are cells of theimmune system, such as cells of the innate or adaptive immunity, e.g.,myeloid or lymphoid cells, including lymphocytes, typically T cellsand/or NK cells. Other exemplary cells include stem cells, such asmultipotent and pluripotent stem cells, including induced pluripotentstem cells (iPSCs). The cells typically are primary cells, such as thoseisolated directly from a subject and/or isolated from a subject andfrozen. In some embodiments, the cells include one or more subsets of Tcells or other cell types, such as whole T cell populations, CD4+ Tcells, CD8+ T cells, and subpopulations thereof, such as those definedby function, activation state, maturity, potential for differentiation,expansion, recirculation, localization, and/or persistence capacities,antigen-specificity, type of antigen receptor, presence in a particularorgan or compartment, marker or cytokine secretion profile, and/ordegree of differentiation. With reference to the subject to be treated,the cells may be allogeneic and/or autologous. Among the methods includeoff-the-shelf methods. In some aspects, such as for off-the-shelftechnologies, the cells are pluripotent and/or multipotent, such as stemcells, such as induced pluripotent stem cells (iPSCs). In someembodiments, the methods include isolating cells from the subject,preparing, processing, culturing, and/or engineering them, andre-introducing them into the same subject, before or aftercryopreservation.

Among the sub-types and subpopulations of T cells and/or of CD4+ and/orof CD8+ T cells are naïve T (TN) cells, effector T cells (TEFF), memoryT cells and sub-types thereof, such as stem cell memory T (TSCM),central memory T (TCM), effector memory T (TEM), or terminallydifferentiated effector memory T cells, tumor-infiltrating lymphocytes(TIL), immature T cells, mature T cells, helper T cells, cytotoxic Tcells, mucosa-associated invariant T (MATT) cells, naturally occurringand adaptive regulatory T (Treg) cells, helper T cells, such as TH1cells, TH2 cells, TH3 cells, TH17 cells, TH9 cells, TH22 cells,follicular helper T cells, alpha/beta T cells, and delta/gamma T cells.

In some embodiments, the cells are natural killer (NK) cells. In someembodiments, the cells are monocytes or granulocytes, e.g., myeloidcells, macrophages, neutrophils, dendritic cells, mast cells,eosinophils, and/or basophils.

In some embodiments, the cells include one or more nucleic acidsintroduced via genetic engineering, and thereby express recombinant orgenetically engineered products of such nucleic acids. In someembodiments, the nucleic acids are heterologous, i.e., normally notpresent in a cell or sample obtained from the cell, such as one obtainedfrom another organism or cell, which for example, is not ordinarilyfound in the cell being engineered and/or an organism from which suchcell is derived. In some embodiments, the nucleic acids are notnaturally occurring, such as a nucleic acid not found in nature,including one comprising chimeric combinations of nucleic acids encodingvarious domains from multiple different cell types.

In some embodiments, preparation of the engineered cells includes one ormore culture and/or preparation steps. The cells for introduction of thenucleic acid encoding the transgenic receptor such as the CAR, may beisolated from a sample, such as a biological sample, e.g., one obtainedfrom or derived from a subject. In some embodiments, the subject fromwhich the cell is isolated is one having the disease or condition or inneed of a cell therapy or to which cell therapy will be administered.The subject in some embodiments is a human in need of a particulartherapeutic intervention, such as the adoptive cell therapy for whichcells are being isolated, processed, and/or engineered.

Accordingly, the cells in some embodiments are primary cells, e.g.,primary human cells. The samples include tissue, fluid, and othersamples taken directly from the subject, as well as samples resultingfrom one or more processing steps, such as separation, centrifugation,genetic engineering (e.g. transduction with viral vector), washing,and/or incubation. The biological sample can be a sample obtaineddirectly from a biological source or a sample that is processed.Biological samples include, but are not limited to, body fluids, such asblood, plasma, serum, cerebrospinal fluid, synovial fluid, urine andsweat, tissue and organ samples, including processed samples derivedtherefrom.

In some aspects, the sample from which the cells are derived or isolatedis blood or a blood-derived sample, or is or is derived from anapheresis or leukapheresis product. Exemplary samples include wholeblood, peripheral blood mononuclear cells (PBMCs), leukocytes, bonemarrow, thymus, tissue biopsy, tumor, leukemia, lymphoma, lymph node,gut associated lymphoid tissue, mucosa associated lymphoid tissue,spleen, other lymphoid tissues, liver, lung, stomach, intestine, colon,kidney, pancreas, breast, bone, prostate, cervix, testes, ovaries,tonsil, or other organ, and/or cells derived therefrom. Samples include,in the context of cell therapy, e.g., adoptive cell therapy, samplesfrom autologous and allogeneic sources.

In some embodiments, the cells are derived from cell lines, e.g., T celllines. The cells in some embodiments are obtained from a xenogeneicsource, for example, from mouse, rat, non-human primate, and pig.

In some embodiments, isolation of the cells includes one or morepreparation and/or non-affinity based cell separation steps. In someexamples, cells are washed, centrifuged, and/or incubated in thepresence of one or more reagents, for example, to remove unwantedcomponents, enrich for desired components, lyse or remove cellssensitive to particular reagents. In some examples, cells are separatedbased on one or more property, such as density, adherent properties,size, sensitivity and/or resistance to particular components.

In some examples, cells from the circulating blood of a subject areobtained, e.g., by apheresis or leukapheresis. The samples, in someaspects, contain lymphocytes, including T cells, monocytes,granulocytes, B cells, other nucleated white blood cells, red bloodcells, and/or platelets, and in some aspects contains cells other thanred blood cells and platelets.

In some embodiments, the blood cells collected from the subject arewashed, e.g., to remove the plasma fraction and to place the cells in anappropriate buffer or media for subsequent processing steps. In someembodiments, the cells are washed with phosphate buffered saline (PBS).In some embodiments, the wash solution lacks calcium and/or magnesiumand/or many or all divalent cations. In some aspects, a washing step isaccomplished a semi-automated “flow-through” centrifuge (for example,the Cobe 2991 cell processor, Baxter) according to the manufacturer'sinstructions. In some aspects, a washing step is accomplished bytangential flow filtration (TFF) according to the manufacturer'sinstructions. In some embodiments, the cells are resuspended in avariety of biocompatible buffers after washing, such as, for example,Ca++/Mg++ free PBS. In certain embodiments, components of a blood cellsample are removed and the cells directly resuspended in culture media.

In some embodiments, the methods include density-based cell separationmethods, such as the preparation of white blood cells from peripheralblood by lysing the red blood cells and centrifugation through a Percollor Ficoll gradient.

In some embodiments, the isolation methods include the separation ofdifferent cell types based on the expression or presence in the cell ofone or more specific molecules, such as surface markers, e.g., surfaceproteins, intracellular markers, or nucleic acid. In some embodiments,any known method for separation based on such markers may be used. Insome embodiments, the separation is affinity- or immunoaffinity-basedseparation. For example, the isolation in some aspects includesseparation of cells and cell populations based on the cells' expressionor expression level of one or more markers, typically cell surfacemarkers, for example, by incubation with an antibody or binding partnerthat specifically binds to such markers, followed generally by washingsteps and separation of cells having bound the antibody or bindingpartner, from those cells having not bound to the antibody or bindingpartner.

Such separation steps can be based on positive selection, in which thecells having bound the reagents are retained for further use, and/ornegative selection, in which the cells having not bound to the antibodyor binding partner are retained. In some examples, both fractions areretained for further use. In some aspects, negative selection can beparticularly useful where no antibody is available that specificallyidentifies a cell type in a heterogeneous population, such thatseparation is best carried out based on markers expressed by cells otherthan the desired population.

The separation need not result in 100% enrichment or removal of aparticular cell population or cells expressing a particular marker. Forexample, positive selection of or enrichment for cells of a particulartype, such as those expressing a marker, refers to increasing the numberor percentage of such cells, but need not result in a complete absenceof cells not expressing the marker. Likewise, negative selection,removal, or depletion of cells of a particular type, such as thoseexpressing a marker, refers to decreasing the number or percentage ofsuch cells, but need not result in a complete removal of all such cells.

In some examples, multiple rounds of separation steps are carried out,where the positively or negatively selected fraction from one step issubjected to another separation step, such as a subsequent positive ornegative selection. In some examples, a single separation step candeplete cells expressing multiple markers simultaneously, such as byincubating cells with a plurality of antibodies or binding partners,each specific for a marker targeted for negative selection. Likewise,multiple cell types can simultaneously be positively selected byincubating cells with a plurality of antibodies or binding partnersexpressed on the various cell types.

For example, in some aspects, specific subpopulations of T cells, suchas cells positive or expressing high levels of one or more surfacemarkers, e.g., CD28+, CD62L+, CCR7+, CD27+, CD127+, CD4+, CD8+, CD45RA+,and/or CD45RO+ T cells, are isolated by positive or negative selectiontechniques.

For example, CD3+, CD28+ T cells can be positively selected usingCD3/CD28 conjugated magnetic beads (e.g., DYNABEADS® M-450 CD3/CD28 TCell Expander).

In some embodiments, isolation is carried out by enrichment for aparticular cell population by positive selection, or depletion of aparticular cell population, by negative selection. In some embodiments,positive or negative selection is accomplished by incubating cells withone or more antibodies or other binding agent that specifically bind toone or more surface markers expressed or expressed (marker+) at arelatively higher level (marker^(high)) on the positively or negativelyselected cells, respectively.

In some embodiments, T cells are separated from a PBMC sample bynegative selection of markers expressed on non-T cells, such as B cells,monocytes, or other white blood cells, such as CD14. In some aspects, aCD4+ or CD8+ selection step is used to separate CD4+ helper and CD8+cytotoxic T cells. Such CD4+ and CD8+ populations can be further sortedinto sub-populations by positive or negative selection for markersexpressed or expressed to a relatively higher degree on one or morenaive, memory, and/or effector T cell subpopulations.

In some embodiments, CD8+ T cells are further enriched for or depletedof naive, central memory, effector memory, and/or central memory stemcells, such as by positive or negative selection based on surfaceantigens associated with the respective subpopulation. In someembodiments, enrichment for central memory T (TCM) cells is carried outto increase efficacy, such as to improve long-term survival, expansion,and/or engraftment following administration, which in some aspects isparticularly robust in such sub-populations. See Terakura et al. Blood.1:72-82 (2012); Wang et al. J Immunother. 35(9):689-701 (2012). In someembodiments, combining TCM-enriched CD8+ T cells and CD4+ T cellsfurther enhances efficacy.

In embodiments, memory T cells are present in both CD62L+ and CD62L-subsets of CD8+ peripheral blood lymphocytes. PBMC can be enriched foror depleted of CD62L-CD8+ and/or CD62L+CD8+ fractions, such as usinganti-CD8 and anti-CD62L antibodies.

In some embodiments, the enrichment for central memory T (TCM) cells isbased on positive or high surface expression of CD45RO, CD62L, CCR7,CD28, CD3, and/or CD 127; in some aspects, it is based on negativeselection for cells expressing or highly expressing CD45RA and/orgranzyme B. In some aspects, isolation of a CD8+ population enriched forTCM cells is carried out by depletion of cells expressing CD4, CD14,CD45RA, and positive selection or enrichment for cells expressing CD62L.In one aspect, enrichment for central memory T (TCM) cells is carriedout starting with a negative fraction of cells selected based on CD4expression, which is subjected to a negative selection based onexpression of CD14 and CD45RA, and a positive selection based on CD62L.Such selections in some aspects are carried out simultaneously and inother aspects are carried out sequentially, in either order. In someaspects, the same CD4 expression-based selection step used in preparingthe CD8+ T cell population or subpopulation, also is used to generatethe CD4+ T cell population or sub-population, such that both thepositive and negative fractions from the CD4-based separation areretained and used in subsequent steps of the methods, optionallyfollowing one or more further positive or negative selection steps.

In a particular example, a sample of PBMCs or other white blood cellsample is subjected to selection of CD4+ T cells, where both thenegative and positive fractions are retained. The negative fraction thenis subjected to negative selection based on expression of CD14 andCD45RA or CD19, and positive selection based on a marker characteristicof central memory T cells, such as CD62L or CCR7, where the positive andnegative selections are carried out in either order.

CD4+ T helper cells are sorted into naïve, central memory, and effectorcells by identifying cell populations that have cell surface antigens.CD4+ lymphocytes can be obtained by standard methods. In someembodiments, naive CD4+ T lymphocytes are CD45RO−, CD45RA+, CD62L+, CD4+T cells. In some embodiments, central memory CD4+ T cells are CD62L+ andCD45RO+. In some embodiments, effector CD4+ T cells are CD62L- andCD45RO−.

In one example, to enrich for CD4+ T cells by negative selection, amonoclonal antibody cocktail typically includes antibodies to CD14,CD20, CD11b, CD16, HLA-DR, and CD8. In some embodiments, the antibody orbinding partner is bound to a solid support or matrix, such as amagnetic bead or paramagnetic bead, to allow for separation of cells forpositive and/or negative selection. For example, in some embodiments,the cells and cell populations are separated or isolated usingimmunomagnetic (or affinitymagnetic) separation techniques (reviewed inMethods in Molecular Medicine, vol. 58: Metastasis Research Protocols,Vol. 2: Cell Behavior In Vitro and In Vivo, p 17-25 Edited by: S. A.Brooks and U. Schumacher© Humana Press Inc., Totowa, N.J.).

In some aspects, the sample or composition of cells to be separated isincubated with small, magnetizable or magnetically responsive material,such as magnetically responsive particles or microparticles, such asparamagnetic beads (e.g., such as Dynalbeads or MACS beads). Themagnetically responsive material, e.g., particle, generally is directlyor indirectly attached to a binding partner, e.g., an antibody, thatspecifically binds to a molecule, e.g., surface marker, present on thecell, cells, or population of cells that it is desired to separate,e.g., that it is desired to negatively or positively select.

In some embodiments, the magnetic particle or bead comprises amagnetically responsive material bound to a specific binding member,such as an antibody or other binding partner. There are many well-knownmagnetically responsive materials used in magnetic separation methods.Suitable magnetic particles include those described in Molday, U.S. Pat.No. 4,452,773, and in European Patent Specification EP 452342 B, whichare hereby incorporated by reference. Colloidal sized particles, such asthose described in Owen U.S. Pat. No. 4,795,698, and Liberti et al.,U.S. Pat. No. 5,200,084 are other examples.

The incubation generally is carried out under conditions whereby theantibodies or binding partners, or molecules, such as secondaryantibodies or other reagents, which specifically bind to such antibodiesor binding partners, which are attached to the magnetic particle orbead, specifically bind to cell surface molecules if present on cellswithin the sample.

In some aspects, the sample is placed in a magnetic field, and thosecells having magnetically responsive or magnetizable particles attachedthereto will be attracted to the magnet and separated from the unlabeledcells. For positive selection, cells that are attracted to the magnetare retained; for negative selection, cells that are not attracted(unlabeled cells) are retained. In some aspects, a combination ofpositive and negative selection is performed during the same selectionstep, where the positive and negative fractions are retained and furtherprocessed or subject to further separation steps.

In certain embodiments, the magnetically responsive particles are coatedin primary antibodies or other binding partners, secondary antibodies,lectins, enzymes, or streptavidin. In certain embodiments, the magneticparticles are attached to cells via a coating of primary antibodiesspecific for one or more markers. In certain embodiments, the cells,rather than the beads, are labeled with a primary antibody or bindingpartner, and then cell-type specific secondary antibody- or otherbinding partner (e.g., streptavidin)-coated magnetic particles, areadded. In certain embodiments, streptavidin-coated magnetic particlesare used in conjunction with biotinylated primary or secondaryantibodies.

In some embodiments, the magnetically responsive particles are leftattached to the cells that are to be subsequently incubated, culturedand/or engineered; in some aspects, the particles are left attached tothe cells for administration to a patient. In some embodiments, themagnetizable or magnetically responsive particles are removed from thecells. Methods for removing magnetizable particles from cells are knownand include, e.g., the use of competing non-labeled antibodies, andmagnetizable particles or antibodies conjugated to cleavable linkers. Insome embodiments, the magnetizable particles are biodegradable.

In some embodiments, the affinity-based selection is viamagnetic-activated cell sorting (MACS) (Miltenyi Biotec, Auburn,Calif.). Magnetic Activated Cell Sorting (MACS) systems are capable ofhigh-purity selection of cells having magnetized particles attachedthereto. In certain embodiments, MACS operates in a mode wherein thenon-target and target species are sequentially eluted after theapplication of the external magnetic field. That is, the cells attachedto magnetized particles are held in place while the unattached speciesare eluted. Then, after this first elution step is completed, thespecies that were trapped in the magnetic field and were prevented frombeing eluted are freed in some manner such that they can be eluted andrecovered. In certain embodiments, the non-target cells are labelled anddepleted from the heterogeneous population of cells.

In certain embodiments, the isolation or separation is carried out usinga system, device, or apparatus that carries out one or more of theisolation, cell preparation, separation, processing, incubation,culture, and/or formulation steps of the methods. In some aspects, thesystem is used to carry out each of these steps in a closed or sterileenvironment, for example, to minimize error, user handling and/orcontamination. In one example, the system is a system as described inInternational Patent Application, Publication Number WO2009/072003, orUS 20110003380 A1.

In some embodiments, the system or apparatus carries out one or more,e.g., all, of the isolation, processing, engineering, and formulationsteps in an integrated or self-contained system, and/or in an automatedor programmable fashion. In some aspects, the system or apparatusincludes a computer and/or computer program in communication with thesystem or apparatus, which allows a user to program, control, assess theoutcome of, and/or adjust various aspects of the processing, isolation,engineering, and formulation steps.

In some aspects, the separation and/or other steps is carried out usingCliniMACS system (Miltenyi Biotec), for example, for automatedseparation of cells on a clinical-scale level in a closed and sterilesystem. Components can include an integrated microcomputer, magneticseparation unit, peristaltic pump, and various pinch valves. Theintegrated computer in some aspects controls all components of theinstrument and directs the system to perform repeated procedures in astandardized sequence. The magnetic separation unit in some aspectsincludes a movable permanent magnet and a holder for the selectioncolumn. The peristaltic pump controls the flow rate throughout thetubing set and, together with the pinch valves, ensures the controlledflow of buffer through the system and continual suspension of cells.

The CliniMACS system in some aspects uses antibody-coupled magnetizableparticles that are supplied in a sterile, non-pyrogenic solution. Insome embodiments, after labelling of cells with magnetic particles thecells are washed to remove excess particles. A cell preparation bag isthen connected to the tubing set, which in turn is connected to a bagcontaining buffer and a cell collection bag. The tubing set consists ofpre-assembled sterile tubing, including a pre-column and a separationcolumn, and are for single use only. After initiation of the separationprogram, the system automatically applies the cell sample onto theseparation column. Labelled cells are retained within the column, whileunlabeled cells are removed by a series of washing steps. In someembodiments, the cell populations for use with the methods describedherein are unlabeled and are not retained in the column. In someembodiments, the cell populations for use with the methods describedherein are labeled and are retained in the column. In some embodiments,the cell populations for use with the methods described herein areeluted from the column after removal of the magnetic field, and arecollected within the cell collection bag.

In certain embodiments, separation and/or other steps are carried outusing the CliniMACS Prodigy system (Miltenyi Biotec). The CliniMACSProdigy system in some aspects is equipped with a cell processing unitythat permits automated washing and fractionation of cells bycentrifugation. The CliniMACS Prodigy system can also include an onboardcamera and image recognition software that determines the optimal cellfractionation endpoint by discerning the macroscopic layers of thesource cell product. For example, peripheral blood is automaticallyseparated into erythrocytes, white blood cells and plasma layers. TheCliniMACS Prodigy system can also include an integrated cell cultivationchamber which accomplishes cell culture protocols such as, e.g., celldifferentiation and expansion, antigen loading, and long-term cellculture. Input ports can allow for the sterile removal and replenishmentof media and cells can be monitored using an integrated microscope. See,e.g., Klebanoff et al. J Immunother. 35(9): 651-660 (2012), Terakura etal. Blood. 1:72-82 (2012), and Wang et al. J Immunother. 35(9):689-701(2012).

In some embodiments, a cell population described herein is collected andenriched (or depleted) via flow cytometry, in which cells stained formultiple cell surface markers are carried in a fluidic stream. In someembodiments, a cell population described herein is collected andenriched (or depleted) via preparative scale (FACS)-sorting. In certainembodiments, a cell population described herein is collected andenriched (or depleted) by use of microelectromechanical systems (MEMS)chips in combination with a FACS-based detection system (see, e.g., WO2010/033140, Cho et al. Lab Chip 10, 1567-1573 (2010); and Godin et al.J Biophoton. 1(5):355-376 (2008). In both cases, cells can be labeledwith multiple markers, allowing for the isolation of well-defined T cellsubsets at high purity.

In some embodiments, the antibodies or binding partners are labeled withone or more detectable marker, to facilitate separation for positiveand/or negative selection. For example, separation may be based onbinding to fluorescently labeled antibodies. In some examples,separation of cells based on binding of antibodies or other bindingpartners specific for one or more cell surface markers are carried in afluidic stream, such as by fluorescence-activated cell sorting (FACS),including preparative scale (FACS) and/or microelectromechanical systems(MEMS) chips, e.g., in combination with a flow-cytometric detectionsystem. Such methods allow for positive and negative selection based onmultiple markers simultaneously.

In some embodiments, the preparation methods include steps for freezing,e.g., cryopreserving, the cells, either before or after isolation,incubation, and/or engineering. In some embodiments, the freeze andsubsequent thaw step removes granulocytes and, to some extent, monocytesin the cell population. In some embodiments, the cells are suspended ina freezing solution, e.g., following a washing step to remove plasma andplatelets. Any of a variety of known freezing solutions and parametersin some aspects may be used. One example involves using PBS containing20% DMSO and 8% human serum albumin (HSA), or other suitable cellfreezing media. This is then diluted 1:1 with media so that the finalconcentration of DMSO and HSA are 10% and 4%, respectively. The cellsare generally then frozen to −80° C. at a rate of 1° per minute andstored in the vapor phase of a liquid nitrogen storage tank.

In some embodiments, the cells are incubated and/or cultured prior to orin connection with genetic engineering. The incubation steps can includeculture, cultivation, stimulation, activation, and/or propagation. Theincubation and/or engineering may be carried out in a culture vessel,such as a unit, chamber, well, column, tube, tubing set, valve, vial,culture dish, bag, or other container for culture or cultivating cells.In some embodiments, the compositions or cells are incubated in thepresence of stimulating conditions or a stimulatory agent. Suchconditions include those designed to induce proliferation, expansion,activation, and/or survival of cells in the population, to mimic antigenexposure, and/or to prime the cells for genetic engineering, such as forthe introduction of a recombinant antigen receptor.

The conditions can include one or more of particular media, temperature,oxygen content, carbon dioxide content, time, agents, e.g., nutrients,amino acids, antibiotics, ions, and/or stimulatory factors, such ascytokines, chemokines, antigens, binding partners, fusion proteins,recombinant soluble receptors, and any other agents designed to activatethe cells.

In some embodiments, the stimulating conditions or agents include one ormore agent, e.g., ligand, which is capable of activating anintracellular signaling domain of a TCR complex. In some aspects, theagent turns on or initiates TCR/CD3 intracellular signaling cascade in aT cell. Such agents can include antibodies such as those specific for aTCR, e.g. anti-CD3. In some embodiments, the stimulating conditionsinclude one or more agent, e.g. ligand, which is capable of stimulatinga costimulatory receptor, e.g., anti-CD28. In some embodiments, suchagents and/or ligands may be, bound to solid support such as a bead,and/or one or more cytokines. Optionally, the expansion method mayfurther comprise the step of adding anti-CD3 and/or anti CD28 antibodyto the culture medium (e.g., at a concentration of at least about 0.5ng/mL). In some embodiments, the stimulating agents include IL-2, IL-15and/or IL-7. In some aspects, the IL-2 concentration is at least about10 units/mL.

In some aspects, incubation is carried out in accordance with techniquessuch as those described in U.S. Pat. No. 6,040,177 to Riddell et al.,Klebanoff et al. J Immunother. 35(9): 651-660 (2012), Terakura et al.Blood. 1:72-82 (2012), and/or Wang et al. J Immunother. 35(9):689-701(2012).

In some embodiments, the T cells are expanded by adding to aculture-initiating composition feeder cells, such as non-dividingperipheral blood mononuclear cells (PBMC), (e.g., such that theresulting population of cells contains at least about 5, 10, 20, or 40or more PBMC feeder cells for each T lymphocyte in the initialpopulation to be expanded); and incubating the culture (e.g. for a timesufficient to expand the numbers of T cells). In some aspects, thenon-dividing feeder cells can comprise gamma-irradiated PBMC feedercells. In some embodiments, the PBMC are irradiated with gamma rays inthe range of about 3000 to 3600 rads to prevent cell division. In someaspects, the feeder cells are added to culture medium prior to theaddition of the populations of T cells.

In some embodiments, the stimulating conditions include temperaturesuitable for the growth of human T lymphocytes, for example, at leastabout 25 degrees Celsius, generally at least about 30 degrees, andgenerally at or about 37 degrees Celsius. Optionally, the incubation mayfurther comprise adding non-dividing EBV-transformed lymphoblastoidcells (LCL) as feeder cells. LCL can be irradiated with gamma rays inthe range of about 6000 to 10,000 rads. The LCL feeder cells in someaspects is provided in any suitable amount, such as a ratio of LCLfeeder cells to initial T lymphocytes of at least about 10:1.

In embodiments, antigen-specific T cells, such as antigen-specific CD4+and/or CD8+ T cells, are obtained by stimulating naive or antigenspecific T lymphocytes with antigen. For example, antigen-specific Tcell lines or clones can be generated to cytomegalovirus antigens byisolating T cells from infected subjects and stimulating the cells invitro with the same antigen.

2. Vectors and Methods for Genetic Engineering

Various methods for the introduction of genetically engineeredcomponents, e.g., recombinant receptors, e.g., CARs or TCRs, are wellknown. Exemplary methods include those for transfer of nucleic acidsencoding the receptors, including via viral, e.g., retroviral orlentiviral, transduction, transposons, and electroporation. In someembodiments, surface glycan expression is assessed in a composition ofcells that are collected prior to, during, or immediately after thegenetic engineering process. In some embodiments, the surface glycanexpression is assessed and compared among compositions of cells atcorresponding stages of the process to introduce genetically engineeredcomponents. In some embodiments, the compositions are or have beenengineered to express the same recombinant receptor, but by differentmethods of introducing the genetic material.

In some embodiments, gene transfer is accomplished by first stimulatingthe cell, such as by combining it with a stimulus that induces aresponse such as proliferation, survival, and/or activation, e.g., asmeasured by expression of a cytokine or activation marker, followed bytransduction of the activated cells, and expansion in culture to numberssufficient for clinical applications.

In some embodiments, recombinant nucleic acids are transferred intocells using recombinant infectious virus particles, such as, e.g.,vectors derived from simian virus 40 (SV40), adenoviruses,adeno-associated virus (AAV). In some embodiments, recombinant nucleicacids are transferred into T cells using recombinant lentiviral vectorsor retroviral vectors, such as gamma-retroviral vectors (see, e.g.,Koste et al. Gene Therapy doi: 10.1038/gt.2014.25 (2014); Carlens et al.Exp Hematol., 28(10): 1137-46 (2000); Alonso-Camino et al. Mol Ther NuclAcids, 2, e93 (2013); Park et al., Trends Biotechnol., November 29(11):550-557 (2011).

In some embodiments, the retroviral vector has a long terminal repeatsequence (LTR), e.g., a retroviral vector derived from the Moloneymurine leukemia virus (MoMLV), myeloproliferative sarcoma virus (MPSV),murine embryonic stem cell virus (MESV), murine stem cell virus (MSCV),spleen focus forming virus (SFFV), or adeno-associated virus (AAV). Mostretroviral vectors are derived from murine retroviruses. In someembodiments, the retroviruses include those derived from any avian ormammalian cell source. The retroviruses typically are amphotropic,meaning that they are capable of infecting host cells of severalspecies, including humans. In one embodiment, the gene to be expressedreplaces the retroviral gag, pol and/or env sequences. A number ofillustrative retroviral systems have been described (e.g., U.S. Pat.Nos. 5,219,740; 6,207,453; 5,219,740; Miller and Rosman, BioTechniques,7:980-990 (1989); Miller, A. D. Human Gene Therapy, 1:5-14 (1990);Scarpa et al. Virology, 180:849-852 (1991); Burns et al. Proc. Natl.Acad. Sci. USA, 90:8033-8037 (1993); and Boris-Lawrie and Temin, Cur.Opin. Genet. Develop., 3:102-109 (1993).

Methods of lentiviral transduction are known. Exemplary methods aredescribed in, e.g., Wang et al., J. Immunother., 35(9): 689-701 (2012);Cooper et al. Blood. 101:1637-1644 (2003); Verhoeyen et al., Methods MolBiol., 506: 97-114 (2009); and Cavalieri et al., Blood., 102(2): 497-505(2003).

In some embodiments, recombinant nucleic acids are transferred into Tcells via electroporation (see, e.g., Chicaybam et al, PLoS ONE 8(3):e60298 (2013) and Van Tedeloo et al. Gene Therapy 7(16): 1431-1437(2000)). In some embodiments, recombinant nucleic acids are transferredinto T cells via transposition (see, e.g., Manuri et al. Hum Gene Ther21(4): 427-437 (2010); Sharma et al. Molec Ther Nucl Acids 2, e74(2013); and Huang et al. Methods Mol Biol 506: 115-126 (2009)). Othermethods of introducing and expressing genetic material in immune cellsinclude calcium phosphate transfection (e.g., as described in CurrentProtocols in Molecular Biology, John Wiley & Sons, New York. N.Y.),protoplast fusion, cationic liposome-mediated transfection; tungstenparticle-facilitated microparticle bombardment (Johnston, Nature, 346:776-777 (1990)); and strontium phosphate DNA co-precipitation (Brash etal., Mol. Cell Biol., 7: 2031-2034 (1987)).

Other approaches and vectors for transfer of the nucleic acids encodingthe recombinant products are those described, e.g., in internationalpatent application, Publication No.: WO2014055668, and U.S. Pat. No.7,446,190.

In some embodiments, the cells, e.g., T cells, may be transfected eitherduring or after expansion e.g. with a T cell receptor (TCR) or achimeric antigen receptor (CAR). This transfection for the introductionof the gene of the desired receptor can be carried out with any suitableretroviral vector, for example. The genetically modified cell populationcan then be liberated from the initial stimulus (the CD3/CD28 stimulus,for example) and subsequently be stimulated with a second type ofstimulus e.g. via a de novo introduced receptor). This second type ofstimulus may include an antigenic stimulus in form of a peptide/MHCmolecule, the cognate (cross-linking) ligand of the geneticallyintroduced receptor (e.g. natural ligand of a CAR) or any ligand (suchas an antibody) that directly binds within the framework of the newreceptor (e.g. by recognizing constant regions within the receptor).See, for example, Cheadle et al, Methods Mol Biol. 907:645-66 (2012); orBarrett et al., Chimeric Antigen Receptor Therapy for Cancer AnnualReview of Medicine, Vol. 65: 333-347 (2014).

In some cases, a vector may be used that does not require that thecells, e.g., T cells, are activated. In some such instances, the cellsmay be selected and/or transduced prior to activation. Thus, the cellsmay be engineered prior to, or subsequent to culturing of the cells, andin some cases at the same time as or during at least a portion of theculturing.

In some aspects, the cells further are engineered to promote expressionof cytokines or other factors. Among additional nucleic acids, e.g.,genes for introduction are those to improve the efficacy of therapy,such as by promoting viability and/or function of transferred cells;genes to provide a genetic marker for selection and/or evaluation of thecells, such as to assess in vivo survival or localization; genes toimprove safety, for example, by making the cell susceptible to negativeselection in vivo as described by Lupton S. D. et al., Mol. and CellBiol., 11:6 (1991); and Riddell et al., Human Gene Therapy 3:319-338(1992); see also the publications of PCT/US91/08442 and PCT/US94/05601by Lupton et al. describing the use of bifunctional selectable fusiongenes derived from fusing a dominant positive selectable marker with anegative selectable marker. See, e.g., Riddell et al., U.S. Pat. No.6,040,177, at columns 14-17.

3. Compositions and Formulations

In some embodiments, the cells, such as cells genetically engineeredwith a recombinant receptor (e.g. CAR-T cells) are provided ascompositions, including pharmaceutical compositions and formulations,such as unit dose form compositions including the number of cells foradministration in a given dose or fraction thereof. The pharmaceuticalcompositions and formulations generally include one or more optionalpharmaceutically acceptable carrier or excipient. In some embodiments,the composition includes at least one additional therapeutic agent.

In some embodiments, a composition of cells is generated or manufacturedfor the purposes of a cell therapy. In some embodiments, the cellcomposition is a pharmaceutical composition or formulation. Suchcompositions can be used in accord with the provided methods, forexample, to assess their release for use in the prevention or treatmentof diseases, conditions, and disorders, or in detection, diagnostic, andprognostic methods.

The term “pharmaceutical formulation” refers to a preparation which isin such form as to permit the biological activity of an activeingredient contained therein to be effective, and which contains noadditional components which are unacceptably toxic to a subject to whichthe formulation would be administered. In some embodiments, methodsprovided herein may be used to compare surface glycan expression of cellcompositions composed of the same engineered cells, but with differentpharmaceutical formulations.

A “pharmaceutically acceptable carrier” refers to an ingredient in apharmaceutical formulation, other than an active ingredient, which isnontoxic to a subject. A pharmaceutically acceptable carrier includes,but is not limited to, a buffer, excipient, stabilizer, or preservative.In particular embodiments, methods provided herein may be used tocompare surface glycan expression of cell compositions composed of thesame engineered cells, but with different pharmaceutically acceptablecarriers.

In some embodiments, the T cell therapy, such as engineered T cells(e.g. CAR T cells), are formulated with a pharmaceutically acceptablecarrier. In some aspects, the choice of carrier is determined in part bythe particular cell and/or by the method of administration. Accordingly,there are a variety of suitable formulations. For example, thepharmaceutical composition can contain preservatives. Suitablepreservatives may include, for example, methylparaben, propylparaben,sodium benzoate, and benzalkonium chloride. In some aspects, a mixtureof two or more preservatives is used. The preservative or mixturesthereof are typically present in an amount of about 0.0001% to about 2%by weight of the total composition. Carriers are described, e.g., byRemington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).Pharmaceutically acceptable carriers are generally nontoxic torecipients at the dosages and concentrations employed, and include, butare not limited to: buffers such as phosphate, citrate, and otherorganic acids; antioxidants including ascorbic acid and methionine;preservatives (such as octadecyldimethylbenzyl ammonium chloride;hexamethonium chloride; benzalkonium chloride; benzethonium chloride;phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol);low molecular weight (less than about 10 residues) polypeptides;proteins, such as serum albumin, gelatin, or immunoglobulins;hydrophilic polymers such as polyvinylpyrrolidone; amino acids such asglycine, glutamine, asparagine, histidine, arginine, or lysine;monosaccharides, disaccharides, and other carbohydrates includingglucose, mannose, or dextrins; chelating agents such as EDTA; sugarssuch as sucrose, mannitol, trehalose or sorbitol; salt-formingcounter-ions such as sodium; metal complexes (e.g. Zn-proteincomplexes); and/or non-ionic surfactants such as polyethylene glycol(PEG).

Buffering agents in some aspects are included in the compositions.Suitable buffering agents include, for example, citric acid, sodiumcitrate, phosphoric acid, potassium phosphate, and various other acidsand salts. In some aspects, a mixture of two or more buffering agents isused. The buffering agent or mixtures thereof are typically present inan amount of about 0.001% to about 4% by weight of the totalcomposition. Methods for preparing administrable pharmaceuticalcompositions are known. Exemplary methods are described in more detailin, for example, Remington: The Science and Practice of Pharmacy,Lippincott Williams & Wilkins; 21st ed. (May 1, 2005).

The formulations can include aqueous solutions. The formulation orcomposition may also contain more than one active ingredient useful forthe particular indication, disease, or condition being prevented ortreated with the cells, including one or more active ingredients wherethe activities are complementary to the cells and/or the respectiveactivities do not adversely affect one another. Such active ingredientsare suitably present in combination in amounts that are effective forthe purpose intended. Thus, in some embodiments, the pharmaceuticalcomposition further includes other pharmaceutically active agents ordrugs, such as chemotherapeutic agents, e.g., asparaginase, busulfan,carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil,gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab,vinblastine, vincristine, etc.

The pharmaceutical composition in some embodiments contain cells inamounts effective to treat or prevent the disease or condition, such asa therapeutically effective or prophylactically effective amount.Therapeutic or prophylactic efficacy in some embodiments is monitored byperiodic assessment of treated subjects. For repeated administrationsover several days or longer, depending on the condition, the treatmentis repeated until a desired suppression of disease symptoms occurs.However, other dosage regimens may be useful and can be determined. Thedesired dosage can be delivered by a single bolus administration of thecomposition, by multiple bolus administrations of the composition, or bycontinuous infusion administration of the composition.

The cells may be formulated for administration using standardadministration techniques, formulations, and/or devices. Provided areformulations and devices, such as syringes and vials, for storage andadministration of the compositions. With respect to cells,administration can be autologous or heterologous. For example,immunoresponsive cells or progenitors can be obtained from one subject,and administered to the same subject or a different, compatible subject.Peripheral blood derived immunoresponsive cells or their progeny (e.g.,in vivo, ex vivo or in vitro derived) can be administered via localizedinjection, including catheter administration, systemic injection,localized injection, intravenous injection, or parenteraladministration. When administering a therapeutic composition (e.g., apharmaceutical composition containing a genetically modifiedimmunoresponsive cell), it will generally be formulated in a unit dosageinjectable form (solution, suspension, emulsion).

Formulations include those for oral, intravenous, intraperitoneal,subcutaneous, pulmonary, transdermal, intramuscular, intranasal, buccal,sublingual, or suppository administration. In some embodiments, theagent or cell populations are administered parenterally. The term“parenteral,” as used herein, includes intravenous, intramuscular,subcutaneous, rectal, vaginal, and intraperitoneal administration. Insome embodiments, the agent or cell populations are administered to asubject using peripheral systemic delivery by intravenous,intraperitoneal, or subcutaneous injection.

Compositions in some embodiments are provided as sterile liquidpreparations, e.g., isotonic aqueous solutions, suspensions, emulsions,dispersions, or viscous compositions, which may in some aspects bebuffered to a selected pH. Liquid preparations are normally easier toprepare than gels, other viscous compositions, and solid compositions.Additionally, liquid compositions are somewhat more convenient toadminister, especially by injection. Viscous compositions, on the otherhand, can be formulated within the appropriate viscosity range toprovide longer contact periods with specific tissues. Liquid or viscouscompositions can comprise carriers, which can be a solvent or dispersingmedium containing, for example, water, saline, phosphate bufferedsaline, polyol (for example, glycerol, propylene glycol, liquidpolyethylene glycol) and suitable mixtures thereof.

Sterile injectable solutions can be prepared by incorporating the cellsin a solvent, such as in admixture with a suitable carrier, diluent, orexcipient such as sterile water, physiological saline, glucose,dextrose, or the like. The compositions can also be lyophilized. Thecompositions can contain auxiliary substances such as wetting,dispersing, or emulsifying agents (e.g., methylcellulose), pH bufferingagents, gelling or viscosity enhancing additives, preservatives,flavoring agents, colors, and the like, depending upon the route ofadministration and the preparation desired. Standard texts may in someaspects be consulted to prepare suitable preparations.

Various additives which enhance the stability and sterility of thecompositions, including antimicrobial preservatives, antioxidants,chelating agents, and buffers, can be added. Prevention of the action ofmicroorganisms can be ensured by various antibacterial and antifungalagents, for example, parabens, chlorobutanol, phenol, sorbic acid, andthe like. Prolonged absorption of the injectable pharmaceutical form canbe brought about by the use of agents delaying absorption, for example,aluminum monostearate and gelatin.

The formulations to be used for in vivo administration are generallysterile. Sterility may be readily accomplished, e.g., by filtrationthrough sterile filtration membranes.

For the prevention or treatment of disease, the appropriate dosage maydepend on the type of disease to be treated, the type of agent oragents, the type of cells or recombinant receptors, the severity andcourse of the disease, whether the agent or cells are administered forpreventive or therapeutic purposes, previous therapy, the subject'sclinical history and response to the agent or the cells, and thediscretion of the attending physician. The compositions are in someembodiments suitably administered to the subject at one time or over aseries of treatments.

IV. DEFINITIONS

Unless defined otherwise, all terms of art, notations and othertechnical and scientific terms or terminology used herein are intendedto have the same meaning as is commonly understood by one of ordinaryskill in the art to which the claimed subject matter pertains. In somecases, terms with commonly understood meanings are defined herein forclarity and/or for ready reference, and the inclusion of suchdefinitions herein should not necessarily be construed to represent asubstantial difference over what is generally understood in the art.

As used herein, the singular forms “a,” “an,” and “the” include pluralreferents unless the context clearly dictates otherwise. For example,“a” or “an” means “at least one” or “one or more.” It is understood thataspects and variations described herein include “consisting” and/or“consisting essentially of” aspects and variations.

Throughout this disclosure, various aspects of the claimed subjectmatter are presented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of theclaimed subject matter. Accordingly, the description of a range shouldbe considered to have specifically disclosed all the possible sub-rangesas well as individual numerical values within that range. For example,where a range of values is provided, it is understood that eachintervening value, between the upper and lower limit of that range andany other stated or intervening value in that stated range isencompassed within the claimed subject matter. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges, and are also encompassed within the claimed subjectmatter, subject to any specifically excluded limit in the stated range.Where the stated range includes one or both of the limits, rangesexcluding either or both of those included limits are also included inthe claimed subject matter. This applies regardless of the breadth ofthe range.

The term “about” as used herein refers to the usual error range for therespective value readily known to the skilled person in this technicalfield. Reference to “about” a value or parameter herein includes (anddescribes) embodiments that are directed to that value or parameter perse. For example, description referring to “about X” includes descriptionof “X”. In certain embodiments, “about” a stated value refers to a valuewithin ±25%, ±20%, ±10%, ±5%, ±1%, ±0.1%, or ±0.01% of the stated value.

As used herein, a composition refers to any mixture of two or moreproducts, substances, or compounds, including cells. It may be asolution, a suspension, liquid, powder, a paste, aqueous, non-aqueous orany combination thereof.

As used herein, a statement that a cell or population of cells is“positive” for a particular marker refers to the detectable presence onor in the cell of a particular marker, typically a surface marker. Whenreferring to a surface marker, the term refers to the presence ofsurface expression as detected by flow cytometry, for example, bystaining with an antibody that specifically binds to the marker anddetecting said antibody, wherein the staining is detectable by flowcytometry at a level substantially above the staining detected carryingout the same procedure with an isotype-matched control under otherwiseidentical conditions and/or at a level substantially similar to that forcell known to be positive for the marker, and/or at a levelsubstantially higher than that for a cell known to be negative for themarker.

As used herein, a statement that a cell or population of cells is“negative” for a particular marker refers to the absence of substantialdetectable presence on or in the cell of a particular marker, typicallya surface marker. When referring to a surface marker, the term refers tothe absence of surface expression as detected by flow cytometry, forexample, by staining with an antibody that specifically binds to themarker and detecting said antibody, wherein the staining is not detectedby flow cytometry at a level substantially above the staining detectedcarrying out the same procedure with an isotype-matched control underotherwise identical conditions, and/or at a level substantially lowerthan that for cell known to be positive for the marker, and/or at alevel substantially similar as compared to that for a cell known to benegative for the marker.

The term “vector,” as used herein, refers to a nucleic acid moleculecapable of propagating another nucleic acid to which it is linked. Theterm includes the vector as a self-replicating nucleic acid structure aswell as the vector incorporated into the genome of a host cell intowhich it has been introduced. Certain vectors are capable of directingthe expression of nucleic acids to which they are operatively linked.Such vectors are referred to herein as “expression vectors.”

V. EXEMPLARY EMBODIMENTS

Among the provided embodiments are: 1. A method for assessing cellsurface glycans, the method comprising:

(a) incubating a test composition comprising a plurality of cells underconditions to release one or more glycans from the surface of cells inthe test composition, wherein a sample comprising one or more cellsurface glycans is generated; and

(b) determining the presence, absence, identity and/or level of glycanspresent in the sample, thereby assessing the cell surface glycan profileof the sample.

2. A method for assessing cell surface glycans, the method comprisingdetermining the presence, absence, identity and/or level of glycanspresent in a sample, thereby assessing the cell surface glycan profileof the sample, wherein the sample comprises one or more glycans releasedfrom the surface of cells present in a test composition comprising aplurality of cells after incubation of the test composition underconditions to release the one or more glycans.

3. The method of embodiment 1 or embodiment 2, wherein the glycans areN-glycans.

4. The method of any of embodiments 1-3, wherein cells in the test cellcomposition comprise whole or intact cells.

5. The method of any of embodiments 1-4, wherein the cells are livecells.

6. The method of any of embodiments 1-5, wherein:

the test cell composition is not homogenized or sonicated prior to theincubation; and/or

the test cell composition is not incubated with a protease prior to theincubation, optionally wherein the protease is trypsin; and/or

the cells in the test cell composition, prior to or during theincubation, are not contacted with an agent to extract one or more cellsurface or membrane proteins, optionally wherein the agent is adetergent or protease, optionally trypsin; and/or

less than 10% of the cells are lysed and/or ruptured during theincubation.

7. The method of any of embodiments 1-6, wherein the test cellcomposition comprises no more than 5×10⁶ cells.

8. The method of any of embodiments 1-7, wherein the test cellcomposition comprises between 1×10⁶ cells and 5×10⁶ cells, inclusive.

9. The method of any of embodiments 1-8, wherein the test cellcomposition comprises a concentration of no more than 1×10⁸ cells/mL.

10. The method of any of embodiments 1-9, wherein the test cellcomposition comprises a concentration of between 1×10⁵ cells/mL and1×10⁸ cells/mL, inclusive, between 1×10⁶ cells/mL and 5×10⁷ cells/mL,inclusive, or between 5×10⁶ cells/mL and 2.5×10⁷ cells/mL, inclusive.

11. The method of any of embodiments 1-10, wherein the incubation iscarried out in the presence of an N-glycosidase.

12. The method of embodiment 11, wherein the N-glycosidase is a peptideN-glycosidase (PNGase) F.

13. The method of embodiment 12, wherein the PNGase F is recombinant.

14. The method of any of embodiments 1-6, wherein the one or moreglycans are one or more N-glycans, and wherein the method comprises:

-   -   (i) incubating between 1×10⁶ and 5×10⁶ cells from the test        composition with a recombinant PNGase F under conditions to        release the one or more N-glycans from the surface of the cells        of the test composition;    -   (ii) labeling the one or more N-glycans with a detectable label,        optionally a fluorescent label; and    -   (iii) determining the presence, absence, or level of the labeled        N-glycans, thereby assessing the cell surface glycan profile of        the sample.

15. The method of embodiment 14, wherein the test cell compositioncomprises about 1×10⁶ to 2.5×10⁶ cells.

16. The method of embodiment 13 or embodiment 14, wherein the test cellcomposition comprises a concentration of between 1×10⁶ cells/mL and5×10⁷ cells/mL.

17. The method of any of embodiments 12-16, wherein the PNGase Fcomprises a PGNase F of Flavobacterium meningosepticum, or a portion ormutant thereof that is enzymatically active.

18. The method of any of embodiments 12-17, wherein the PNGase Fcomprises the amino acid sequence set forth in SEQ ID NO: 1 or a portionor mutant thereof that is enzymatically active, or an amino acidsequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%. 92%.93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ IDNO:1 or is a portion thereof that is enzymatically active.

19. The method of any of embodiment 12-18, wherein the PNGase Fcomprises the amino acid sequence set forth in SEQ ID NO: 1.

20. The method of any of embodiments 12-19, wherein the PNGase Fcomprises a tag, optionally an affinity tag.

21. The method of embodiment 20, wherein the tag is a poly-histidine(His-tag). 22. The method of any of embodiments 12-21, wherein:

the PNGase F is greater than or greater than about 90%, greater than orgreater than about 92%, greater than or greater than about 95%, orgreater than or greater than about 98% pure; and/or

the PNGase F comprises less than or less than about 10%, less than orless than about 8%, less than or less than about 5%, less than or lessthan about 2% non-PNGase F protein contaminants; and/or

the PNGase F is greater than or greater than about 90%, greater than orgreater than about 92%, greater than or greater than about 95%, orgreater than or greater than about 98% homogeneous, optionally asdetermined by SDS-PAGE and protein staining, optionally Coomasie Bluestaining.

23. The method of any of embodiments 11-22, wherein the N-glycosydase,optionally PNGase F, is in an enzymatically effective amount to releasethe one or more N-glycans from a native or non-denatured glycoprotein orglycoproteins and/or from the cells of the cell composition afterincubation for no more than 12 hours at a temperature between 35° C. and39° C., optionally about 37° C.

24. The method of embodiment 23, wherein the enzymatically effectiveamount is an amount to release the one or more N-glycans afterincubation for no more than 15 minutes to 3 hours or 30 minutes to 2hours, at a temperature between 25° C. and 39° C. or between 35° C. and39° C., each inclusive, optionally about 37° C.

25. The method of embodiment 23 or embodiment 24, wherein theenzymatically effective amount of PNGase F releases greater than 50%,greater than 55%, greater than 60%, greater than 65%, greater than 70%,greater than 75%, greater than 80%, greater than 85%, greater than 90%,greater than 95%, greater than 99% of N-glycans present on theglycoprotein or glycoproteins and/or present on the surface of the cellcomposition.

26. The method of any of embodiments 1-25, wherein the conditions of theincubation are sufficient to effect release of greater than 50%, greaterthan 55%, greater than 60%, greater than 65%, greater than 70%, greaterthan 75%, greater than 80%, greater than 85%, greater than 90%, greaterthan 95%, greater than 99% N-glycans present on the surface of the testcell composition.

27. The method of any of embodiments 11-26, wherein the amount ofN-glycosidase, optionally PNGase F, is 1 unit to 5000 units, 1 unit to1000 units, 1 unit to 500 units, 1 unit to 250 units, 1 unit to 100units, 1 unit to 50 units, 1 unit to 25 units, 25 units to 5000 units,25 units to 1000 units, 25 units to 500 units, 25 units to 250 units, 25units to 100 units, 25 units to 50 units, 50 units to 5000 units, 50units to 1000 units, 50 units to 500 units, 50 units to 250 units, 50units to 100 units, 100 units to 5000 units, 100 units to 1000 units,100 units to 500 units, 100 units to 250 units, 250 units to 5000 units,250 units to 1000 units, 250 units to 500 units, 500 units to 5000units, 500 units to 1000 units, or 1000 units to 5000 units, eachinclusive.

28. The method of any of embodiments 11-27, wherein the amount ofN-glycosidase, optionally PNGase F, is greater than or greater thanabout or is or is about 1 unit, 5 units, 10 units, 15 units, 20 units,25 units, 50 units, 100 units, 250 units, 500 units, 1000 units, 2500units or 5000 units.

29. The method of embodiment 27 or embodiment 28, wherein one unit is anamount of the N-glycosidase, optionally PNGase F, sufficient to catalyzethe deglycosolation of 1 nanomole of denatured Ribonuclease B (RNase B)in 30 minutes at 37° C.

30. The method of embodiment 27 or embodiment 28, wherein 500 units isan amount of the N-glycosidase, optionally PNGase F, sufficient tocatalyze the deglycosylation of 10 μg of Ribonuclease B (RNase B)incubated in 1XPBS for 5-10 minutes at 37° C. or room temperature.

31. The method of any of embodiments 1-30, wherein the incubating thetest composition is for an amount of time that between or between about5 minutes and 12 hours, 30 minutes and 6 hours or 1 hour and 3 hours,each inclusive.

32. The method of any of embodiments 1-31, wherein the incubating thetest composition is for at least or at least about or is or is about 5minutes, about 10 minutes, about 15 minutes, 30 minutes, 1 hour, 2 hours3 hours, 4 hours, 5 hours or 6 hours.

33. The method of any of embodiments 1-32, wherein the incubating thetest composition is for about 30 minutes.

34. The method of any of embodiments 1-33, wherein the incubating thetest composition is at a temperature between 25° C. and 39° C. orbetween 35° C. and 39° C.

35. The method of any of embodiments 1-35, wherein the incubating thetest composition is at a temperature of about 37° C.

36. The method of any of embodiments 1-35, wherein the incubating thetest composition is for about 30 minutes at a temperature of about 37°C.

37. The method of any of embodiments 1-36, wherein, prior to thedetermining the presence, absence, identity and/or level of glycanspresent in a sample, the method further comprises labeling glycans fromthe sample with a detectable label, optionally a fluorescent label.

38. The method of any of embodiments 14-37, wherein the label is afluorescent label and the fluorescent label is or comprises2-aminobenzamide (2-AB), 2-aminobenzoic acid (2-AA), 2-aminopyridine(PA), 2-Aminoacridone (AMAC), 2-aminonaphthalene trisulfonic acid(ANTS), and 1-aminopyrene-3,6,8-trisulfonic acid (APTS),3-(Acetylamino)-6-aminoacridin (AA-Ac), 6-Aminoquinoline (6-AQ),7-Aminomethyl-coumarin (AMC), 2-Amino (6-amido-biotinyl) pyridine (BAP),9-Fluorenylmethoxycarbonyl (FMOC)-hydrazide,1,2-Diamino-4,5-methylenedioxy-benzene (DMB), or o-Phenylenediamine(OPD).

39. The method of any of embodiments 14-38, wherein the fluorescentlabel comprises a

quinolinyl fluorophore.

40. The method of any of embodiments 14-39, wherein the fluorescentlabel comprises a carbamate tagging group.

41. The method of any of embodiments 14-40, wherein the fluorescentlabel comprises a basic tertiary amine.

42. The methods of any of embodiments 14-37 and 39-41, wherein thefluorescent label comprises a carbamate tagging group, a quinolonefluorophore, and a tertiary amine.

43. The method of any of embodiments 1-42, wherein, prior to determiningthe presence, absence, identity and/or level of the one or more glycans,the sample is subjected to glycan purification or enrichment.

44. The method of embodiment 43, wherein glycan purification orenrichment is carried out by solid phase extraction (SPE).

45. The method of any of embodiments 1-44, wherein determining thepresence, absence, identity and/or level of the one or more glycanscomprises subjecting the sample to mass spectrometry.

46. The method of embodiment 45, the mass spectrometry is electrosprayionization mass spectrometry (ESI-MS), turbospray ionization massspectrometry, nanospray ionization mass spectrometry, thermosprayionization mass spectrometry, sonic spray ionization mass spectrometry,surface enhanced laser desorption ionization mass spectrometry(SELDI-MS) and matrix assisted laser desorption/ionization massspectrometry (MALDI-MS).

47. The method of embodiment 45 or 46, wherein the mass spectrometry isMALDI-MS.

48. The method of any of embodiments 1-47, wherein determining thepresence, absence, identity and/or level of glycans comprises subjectingthe sample to liquid chromatography (LC) followed by mass spectrometry.

49. The method of embodiment 48, wherein the liquid chromatography ishigh performance liquid chromatography (HPLC), ultra-high performanceliquid chromatography (UHPLC), or ultra performance liquidchromatography (UPLC).

50. The method of embodiment 48 or embodiment 49, wherein the liquidchromatography is ultra performance liquid chromatography (UPLC).

51. The method of any of embodiments 48-50, wherein the liquidchromatography and mass spectrometry are carried out online.

52. The method of any of embodiments 48-51, wherein the liquidchromatography is selected from normal phase (NP-), reverse phase (RP)and hydrophilic interaction chromatography (HILIC).

53. The method of any of embodiments 48-52, wherein the liquidchromatography is hydrophilic interaction chromatography (HILIC).

54. The method of any of embodiments 45-53, wherein the massspectrometry comprises electrospray ionization mass spectrometry(ESI-MS), turbospray ionization mass spectrometry, nanospray ionizationmass spectrometry, thermospray ionization mass spectrometry or sonicspray ionization mass spectrometry.

55. The method of any of embodiments 45-54, wherein the massspectrometry comprises ESI-MS.

56. The method of any of embodiments 45-55, wherein the massspectrometry comprises tandem mass spectrometry (MS/MS).

57. The method of any of embodiments 45-56, wherein the massspectrometry comprises tandem ESI-mass spectrometry (ESI-MS/MS).

58. The method of any of embodiments 45-57, wherein the massspectrometer that performs the mass spectrometry comprises one or moreof a quadrupole, ion trap, time of flight (TOF), or Fourier transformion cyclotron resonance mass analyzer.

59. The method of embodiment 58, wherein the mass spectrometer comprisesan ion trap mass analyzer that is a three-dimensional quadrupole iontrap, a cylindrical ion trap, a linear quadrupole ion trap, or anOrbitrap mass analyzer.

60. The method of any of embodiments 45-59, wherein the massspectrometer is a quadrupole-Orbitrap mass spectrometer.

61. The method of any of embodiments 1-60, wherein the determining thepresence, absence, identity and/or level of the one or more glycanscomprises analyzing one or more glycan structure or structures forbranching, linkages between monosaccharides and/or location ofmonosaccharides.

62. The method of any of embodiments 1-61, wherein the one or moreglycans comprises high mannose N-glycans, bisected and Sialyl Lewis'N-glycans, and/or N-acetyl lactosamine containing N-glycans.

63. The method of any of embodiments 1-62, wherein the one or moreglycans comprises a fucosylated biantennary complex glycan having noreducing end terminal galactose residues, a fucosylated biantennarycomplex glycan having one reducing end terminal galactose residue, afucosylated biantennary complex glycan having two reducing end terminalgalactose residues, a biantennary complex glycan having no reducing endterminal galactose residues, a biantennary complex glycan having onereducing end terminal galactose residue, a biantennary complex glycanhaving two reducing end terminal galactose residues, a fucosylatedbiantennary complex glycan having two galactose residues and oneN-acetylneuraminic acid residue, a fucosylated biantennary complexglycan having two galactose residues and two N-acetylneuraminic acidresidues, a biantennary complex glycan having two galactose residues andtwo N-acetylneuraminic acid residues, a high mannose glycan having fivemannose residues, a high mannose glycan having six mannose residues, ahigh mannose glycan having seven mannose residues, a high mannose glycanhaving eight mannose residues, and/or a high mannose glycan having ninemannose residues.

64. The methods of any of embodiments 1-63, wherein the determining thepresence, absence, identity and/or level of glycans present in thesample comprises determining the presence, absence, identity and/orlevel of at least 25, at least 50, at least 60, at least 70, at least80, at least 90, at least 100, or at least 200 different species ofglycans.

65. The method of any of embodiments 1-64, wherein the presence or levelof a glycan species present in the sample is determined if at least 100amol, at least 500 amol, at least 1 fmol, at least 5 fmol, or at least10 fmol of the glycan species is present in the sample.

66. The method of any of embodiments 1-65, wherein the presence or levelof a glycan species present in the sample is determined if glycanspecies makes up at least 0.00001%, 0.00005%, 0.0001%, 0.0005%, 0.001%,0.005%, or 0.01% of the total glycans in the sample.

67. The method of any of embodiments 62-65, wherein the species ofglycans are species of N-glycans.

68. The method of any of embodiments 1-67, wherein the test cellcomposition is or comprises at least a portion of a source cellcomposition comprising the plurality of cells.

69. The method of any of embodiments 1-68, wherein the cells comprisemammalian cells or the test cell composition comprises mammalian cells.

70. The method of any one of embodiments 1-69, wherein the cellscomprise human cells or the test cell composition comprises human cells.

71. The method of any one of embodiments 1-70, wherein the cellscomprise stem cells or the test cell composition comprises stem cells.

72. The method of embodiment 71, wherein the stem cell is an inducedpluripotent stem cell (iPSC).

73. The method of any of embodiments 1-70, wherein the test cellcomposition comprises cells present in an apheresis product or aleukapheresis product or cells derived therefrom.

74. The method of any of embodiments 1-70, and 73, wherein:

-   -   the cells comprise immune cells, white blood cells, peripheral        blood mononuclear cells (PBMC), lymphocytes, or unfractionated T        cells; or    -   the test cell composition comprises immune cells, white blood        cells, peripheral blood mononuclear cells (PBMC), lymphocytes,        or unfractionated T cells.

75. The method of any one of embodiments 1-74, wherein the cellscomprise an immune cell or the test cell composition comprises immunecells.

76. The method of embodiment 75, wherein the immune cell is a T cell, Bcell, macrophage, neutrophil, natural killer (NK) cell or dendriticcell.

77. The method of any of embodiments 1-76, wherein the cells comprise Tcells that are CD4+ and/or CD8+ T cells or the test cell compositioncomprises T cells that are CD4+ and/or CD8+ T cells.

78. The method of any of embodiments 1-77, wherein the test cellcomposition comprises:

cells isolated from a biological sample by immunoaffinity-based methods;and/or

cells transduced with a viral vector encoding a recombinant protein;and/or

cell incubated in the presence of one or more test agents, optionallyone more peptide, protein, polypeptide, nucleic acid, small molecule;and/or

cells activated and/or expanded in the presence of one or morestimulating conditions; and/or

cryopreserved cells and/or cells comprising a cryoprotectant; and/or

cells formulated for administration to a subject, optionally in thepresence of a pharmaceutically acceptable excipient.

79. The method of embodiment 78, wherein the test agent is a candidatefor modulating the growth, proliferation, viability, differentiation,intracellular signaling, activation and/or expansion of one or morecells in the test cell composition.

80. The method of embodiment 78 or embodiment 79, wherein thestimulating condition comprises incubation with a stimulatory reagentcapable of activating one or more intracellular signaling domains of oneor more components of a TCR complex and/or one or more intracellularsignaling domains of one or more costimulatory molecules.

81. The method of embodiment 80, wherein the stimulatory reagentcomprises a primary agent that specifically binds to a member of a TCRcomplex and a secondary agent that specifically binds to a T cellcostimulatory molecule.

82. The method of embodiment 81, wherein the primary agent specificallybinds to CD3 and/or the costimulatory molecule is selected from thegroup consisting of CD28, CD137 (4-1-BB), OX40, or ICOS.

83. The method of any of embodiments 80-82, wherein stimulatory reagentcomprises an anti-CD3 antibody or antigen binding fragment thereof andan anti-CD28 antibody or an antigen-binding fragment thereto.

84. The method of any of embodiments 80-83, wherein the primary andsecondary agents comprise antibodies and/or are present on the surfaceof a solid support.

85. The method of embodiment 84, wherein the solid support is orcomprises a bead. 86. The method of any of embodiments 1-85, wherein thecells express a recombinant receptor or the test composition comprisescells expressing a recombinant receptor.

87. The method of embodiment 86, wherein the recombinant receptor is orcomprises a chimeric receptor and/or a recombinant antigen receptor.

88. The method of embodiment 86 or embodiment 87, wherein therecombinant receptor is capable of binding to a target antigen that isassociated with, specific to, and/or expressed on a cell or tissue of adisease, disorder or condition.

89. The method of embodiment 88, wherein the disease, disorder orcondition is an infectious disease or disorder, an autoimmune disease,an inflammatory disease, or a tumor or a cancer.

90. The method of embodiment 88 or embodiment 89, wherein the targetantigen is a tumor antigen.

91. The method of any of embodiments 88-90, wherein the target antigenis selected from among ROR1, B cell maturation antigen (BCMA), carbonicanhydrase 9 (CAIX), tEGFR, Her2/neu (receptor tyrosine kinase erbB2),L1-CAM, CD19, CD20, CD22, mesothelin, CEA, and hepatitis B surfaceantigen, anti-folate receptor, CD23, CD24, CD30, CD33, CD38, CD44, EGFR,epithelial glycoprotein 2 (EPG-2), epithelial glycoprotein 40 (EPG-40),EPHa2, erb-B2, erb-B3, erb-B4, erbB dimers, EGFR vIII, folate bindingprotein (FBP), FCRLS, FCRHS, fetal acetylcholine receptor, GD2, GD3,HMW-MAA, IL-22R-alpha, IL-13R-alpha2, kinase insert domain receptor(kdr), kappa light chain, Lewis Y, L1-cell adhesion molecule, (L1-CAM),Melanoma-associated antigen (MAGE)-A1, MAGE-A3, MAGE-A6, Preferentiallyexpressed antigen of melanoma (PRAME), survivin, TAG72, B7-H6, IL-13receptor alpha 2 (IL-13Ra2), CA9, GD3, HMW-MAA, CD171, G250/CAIX, HLA-AIMAGE A1, HLA-A2 NY-ESO-1, PSCA, folate receptor-a, CD44v6, CD44v7/8,avb6 integrin, 8H9, NCAM, VEGF receptors, 5T4, Foetal AchR, NKG2Dligands, CD44v6, dual antigen, a cancer-testes antigen, mesothelin,murine CMV, mucin 1 (MUC1), MUC16, PSCA, NKG2D, NY-ESO-1, MART-1, gp100,oncofetal antigen, ROR1, TAG72, VEGF-R2, carcinoembryonic antigen (CEA),Her2/neu, estrogen receptor, progesterone receptor, ephrinB2, CD123,c-Met, GD-2, O-acetylated GD2 (OGD2), CE7, Wilms Tumor 1 (WT-1), acyclin, cyclin A2, CCL-1, CD138, a pathogen-specific antigen and anantigen associated with a universal tag.

92. The method of any of embodiments 88-91, wherein the recombinantreceptor is or comprises a functional non-TCR antigen receptor or a TCRor antigen-binding fragment thereof 93. The method of any of embodiments88-92, wherein the recombinant receptor is a chimeric antigen receptor(CAR).

94. A method of assaying a cell composition, the method comprising:

(a) assessing the cell surface glycan profile in a sample from a testcell composition comprising a plurality of cells according to the methodof any of embodiments 1-91; and

(b) comparing the cell surface glycan profile of the sample to the cellsurface glycan profile of a reference sample.

95. A method of assaying a cell composition, the method comprisingcomparing the cell surface glycan profile of a sample compared to thecell surface profile of a reference sample, wherein cell surface glycanprofile of the sample is or has been determined according to the methodof any of embodiments 1-93 from a test cell composition comprising aplurality of cells.

96. The method of embodiment 94 or embodiment 95, wherein the cellsurface glycan profile comprises at least 25, at least 50, at least 60,at least 70, at least 80, at least 90, at least 100, or at least 200different species of glycans, optionally different species of N-glycans.

97. The method of any of embodiments 94-96, wherein the cell surfaceglycan profile comprises high mannose N-glycans, bisected and SialylLewis' N-glycans, and/or N-acetyl lactosamine containing N-glycans.

98. The method of embodiment 94-97, wherein the cell surface glycanprofile comprise a fucosylated biantennary complex glycan having noreducing end terminal galactose residues, a fucosylated biantennarycomplex glycan having one reducing end terminal galactose residue, afucosylated biantennary complex glycan having two reducing end terminalgalactose residues, a biantennary complex glycan having no reducing endterminal galactose residues, a biantennary complex glycan having onereducing end terminal galactose residue, a biantennary complex glycanhaving two reducing end terminal galactose residues, a fucosylatedbiantennary complex glycan having two galactose residues and oneN-acetylneuraminic acid residue, a fucosylated biantennary complexglycan having two galactose residues and two N-acetylneuraminic acidresidues, a biantennary complex glycan having two galactose residues andtwo N-acetylneuraminic acid residues, a high mannose glycan having fivemannose residues, a high mannose glycan having six mannose residues, ahigh mannose glycan having seven mannose residues, a high mannose glycanhaving eight mannose residues, and/or a high mannose glycan having ninemannose residues.

99. The method of any of embodiments 94-98, wherein the cell surfaceglycan profile comprises the glycans in Table E1 or a subset thereof.

100. The method of any of embodiments 94-99, wherein the referencesample is a reference standard comprising a release specification, alabel requirement or a compendia specification.

101. The method of any of embodiments 94-100, wherein the cellcomposition is released for treatment of a subject only if the cellsurface glycan profile of the composition is substantially the same asthe reference sample and/or if the percent of a target glycan or each ofa plurality of target glycans to the total glycans present in the samplediffers by no more than 25%, no more than 20% or no more than 10% fromthe percent of the target glycan or each of the plurality of targetglycans to the total glycans present in the reference sample.

102. The method of embodiment 101, wherein the target glycans compriseat least 25, at least 50, at least 60, at least 70, at least 80, atleast 90, at least 100, or at least 200 different species of glycans.

103. The method of embodiment 101 or embodiment 102, wherein the targetglycan or glycans comprise high mannose N-glycans, bisected and SialylLewis' N-glycans, and/or N-acetyl lactosamine containing N-glycans.

104. The method of any of embodiments 101-103, wherein the target glycanor glycans comprise a fucosylated biantennary complex glycan having noreducing end terminal galactose residues, a fucosylated biantennarycomplex glycan having one reducing end terminal galactose residue, afucosylated biantennary complex glycan having two reducing end terminalgalactose residues, a biantennary complex glycan having no reducing endterminal galactose residues, a biantennary complex glycan having onereducing end terminal galactose residue, a biantennary complex glycanhaving two reducing end terminal galactose residues, a fucosylatedbiantennary complex glycan having two galactose residues and oneN-acetylneuraminic acid residue, a fucosylated biantennary complexglycan having two galactose residues and two N-acetylneuraminic acidresidues, a biantennary complex glycan having two galactose residues andtwo N-acetylneuraminic acid residues, a high mannose glycan having fivemannose residues, a high mannose glycan having six mannose residues, ahigh mannose glycan having seven mannose residues, a high mannose glycanhaving eight mannose residues, and/or a high mannose glycan having ninemannose residues.

105. The method of any of embodiments 101-104, wherein the target glycanor glycans comprise the glycans present in Table E1 or a subset thereof.

106. The method of any of embodiments 101-105, wherein the target glycanor glycans comprise the glycans detectable in the cell surface glycanprofile.

107. The method of any of embodiments 95-99, wherein the referencesample is a cell surface glycan profile from a different cellcomposition.

108. The method of embodiment 107, wherein the different cellcomposition is from a different stage of a manufacturing process forproducing the test cell composition or a source cell composition fromwhich the test composition has been derived or obtained, wherein thestage of the manufacturing process optionally is a prior stage of themanufacturing process.

109. The method of embodiment 108, wherein the manufacturing processcomprises one or more stages selected from:

cells isolated from a biological sample by leukapheresis or apheresis;

cells selected from a biological sample by immunoaffinity-based methods;and/or

cells introduced with a recombinant nucleic acid, optionally a viralvector encoding a recombinant protein; and/or

cell incubated in the presence of one or more test agents, optionallyone more peptide, protein, polypeptide, nucleic acid, small molecule;and/or

cells activated and/or expanded in the presence of one or morestimulating conditions; and/or

cryopreservation of cells in the presence of a cryoprotectant; and/or

cells formulated for administration to a subject, optionally in thepresence of a pharmaceutically acceptable excipient.

110. The method of any of embodiments 107-109, wherein a difference inthe glycan profile between the test composition and reference sampleindicates one or more differences is present in the cells among thecells produced at the different stages in the manufacturing process.

111. The method of embodiment 110, wherein the difference in the glycanprofile exists if the cell surface glycan profile of the composition issubstantially different from the reference sample and/or if the percentof a target glycan or each of the one or more target glycans to thetotal glycans present in the sample differs by greater than or greaterthan about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more from thepercent of the target glycan or each of the one or more target glycansto the total glycans present in the reference sample.

112. The method of embodiment 111, wherein the target glycans compriseat least 25, at least 50, at least 60, at least 70, at least 80, atleast 90, at least 100, or at least 200 different species of glycans,optionally wherein the species of glycans are N-glycans.

113. The method of embodiment 110 or embodiment 112, wherein the targetglycan or glycans comprise high mannose N-glycans, bisected and SialylLewis^(X) N-glycans, and/or N-acetyl lactosamine containing N-glycans.

114. The method of any of embodiments 111-113, wherein the target glycanor glycans comprise a fucosylated biantennary complex glycan having noreducing end terminal galactose residues, a fucosylated biantennarycomplex glycan having one reducing end terminal galactose residue, afucosylated biantennary complex glycan having two reducing end terminalgalactose residues, a biantennary complex glycan having no reducing endterminal galactose residues, a biantennary complex glycan having onereducing end terminal galactose residue, a biantennary complex glycanhaving two reducing end terminal galactose residues, a fucosylatedbiantennary complex glycan having two galactose residues and oneN-acetylneuraminic acid residue, a fucosylated biantennary complexglycan having two galactose residues and two N-acetylneuraminic acidresidues, a biantennary complex glycan having two galactose residues andtwo N-acetylneuraminic acid residues, a high mannose glycan having fivemannose residues, a high mannose glycan having six mannose residues, ahigh mannose glycan having seven mannose residues, a high mannose glycanhaving eight mannose residues, and/or a high mannose glycan having ninemannose residues.

115. The method of any of embodiments 111-114, wherein the target glycanor glycans comprise the glycans present in Table E1 or a subset thereof.

116. The method of any of embodiments 111-115, wherein the target glycanor glycans comprise the glycans detectable in the cell surface glycanprofile.

117. The method of any of embodiments 111-116, wherein the one moredifferences is associated with a functional activity or phenotype of thecells.

118. The method of embodiment 117, wherein the functional activity orphenotype comprises one or more of masking of a cell surface marker, ametabolic activity, differentiation state, proliferative or expansioncapacity, activation state, cytolytic activity, signaling activity, anadhesion property, or a homing property.

119. The method of any of embodiments 111-118, further comprisingmodulating or changing the process for manufacturing the cellcomposition.

120. The method of any of embodiments 108-119, wherein the referencestandard comprises an average or median of the presence, absence,identity and/or level of the one or more target glycan or glycans amonga plurality of compositions produced by the process.

121. A method for manufacturing a cell composition, comprising

incubating and/or contacting an input composition comprising a pluralityof cells with one or more agents and/or under one or more conditionsthereby generating the cell composition,

wherein the cell composition comprises one or a plurality of cells thatare genetically, phenotypically, and/or functionally different from oneor a plurality of cells from the input composition, and

wherein the cell composition comprises one or a plurality of cells thatcomprise a cell surface glycan profile comprising one or more targetglycans and/or each of the one or more target glycans in the cellsurface glycan profile differs by no more than 25% from the cell surfaceglycan profile or each of the one or more target glycans to the totalglycans present in a reference sample, wherein the cell surface glycanprofile comprises glycans released from the surface of cells in the cellcomposition.

122. The method of embodiment 121, wherein the cell surface glycanprofile or the one or more target glycans is determined according to themethod of any of embodiments 1-93.

123. The method of embodiment 121 or embodiment 122, wherein the cellcomposition comprises cells comprising a recombinant nucleic acid.

124. The method of any of embodiments 121-123, wherein prior to, during,or subsequent to the incubation and/or contacting, the method comprisesone or more steps selected from cell washing, dilution, isolation,selection, separation, cultivation, stimulation, introduction of arecombinant nucleic acid, cryopreservation, formulation and/orpackaging.

125. The method of any of embodiments 121-124, wherein prior to, during,or subsequent to incubation and/or contacting, the method comprises oneor more steps selected from:

isolating cells from a biological sample by leukapheresis or apheresis;

isolating cells from a biological sample by immunoaffinity-basedmethods; and/or

introducing cells with a recombinant nucleic acid, optionally a viralvector encoding a recombinant protein; and/or

incubating cells in the presence of one or more agent, optionally onemore peptide, protein, polypeptide, nucleic acid, small molecule; and/or

activating cells and/or expanded in the presence of one or morestimulating conditions; and/or

cryopreserving cells in the presence of a cryoprotectant; and/or

formulating cells for administration to a subject, optionally in thepresence of a pharmaceutically acceptable excipient.

126. The method of any of embodiments 121-125, wherein the one or moreagents or conditions comprises presence or concentration of serum; timein culture; presence or amount of a stimulating agent; the type orextent of a stimulating agent; presence or amount of amino acids;temperature; the source or cell types of the source composition; theratio or percentage of cell types in the source composition, optionallythe CD4+/CD8+ T cell ratio; the presence or amount of beads; celldensity; static culture; rocking culture; perfusion; the type of viralvector; the vector copy number; the presence of a transduction adjuvant;cell density of the source composition in cryopreservation; the extentof expression of the recombinant receptor; or the presence of a compoundto modulate cell phenotype.

127. The method of any of embodiments 121-126, wherein the one or moreagents or conditions comprises stimulating conditions, a peptide, aprotein, a polypeptide, a nucleic acid, a small molecule, and/or arecombinant nucleic acid, optionally a viral vector encoding arecombinant protein.

128. The method of any of embodiments 121-127, wherein the agentmodulates the growth, proliferation, viability, differentiation,intracellular signaling, activation and/or expansion of one or morecells in the cell composition.

129. The method of any of embodiments 125-127, wherein the stimulatingcondition comprises incubation with a stimulatory agent capable ofactivating one or more intracellular signaling domains of one or morecomponents of a TCR complex and/or one or more intracellular signalingdomains of one or more costimulatory molecules.

130. The method of embodiment 129, wherein the stimulatory agentcomprises a primary agent that specifically binds to a member of a TCRcomplex and a secondary agent that specifically binds to a T cellcostimulatory molecule.

131. The method of embodiment 130, wherein the primary agentspecifically binds to CD3 and/or the costimulatory molecule is selectedfrom the group consisting of CD28, CD137 (4-1-BB), OX40, or ICOS.

132. The method of any of embodiments 129-131, wherein stimulatory agentcomprises an anti-CD3 antibody or antigen binding fragment thereofand/or an anti-CD28 antibody or an antigen-binding fragment thereto.133. The method of any of embodiments 129-132, wherein the primary andsecondary agents comprise antibodies and/or are present on the surfaceof a solid support.

134. The method of embodiment 133, wherein the solid support is orcomprises a bead.

135. The method of any of embodiments 125-127 and 129-134, wherein thestimulating conditions comprises the presence of one or more cytokines,optionally IL-2, IL-15 and/or IL-7.

136. The method of any of embodiments 121-135, wherein the referencesample is a reference standard comprising a release specification, alabel requirement or a compendia specification.

137. The method of any of embodiments 121-136, wherein the referencesample comprises an average or median of the presence, absence, identityand/or level of the one or more target glycan or glycans among aplurality of compositions produced by the incubating and/or thecontacting the source composition with the one or more agents and/orunder the one or more conditions.

138. The method of any of embodiments 121-137, wherein the cell surfaceglycan profile comprising the one or more target glycans or each of theone or more target glycans differs by no more than or about 20%, no morethan or about 15%, no more than or about 10% or no more than or about 5%from the cell surface glycan profile or each of the one or more targetglycans to the total glycans present in the reference sample.

139. The method of any of embodiments 121-138, wherein the targetglycans comprise at least 25, at least 50, at least 60, at least 70, atleast 80, at least 90, at least 100, or at least 200 different speciesof glycans, optionally wherein the species of glycans are N-glycans.

140. The method of any of embodiments 121-139, wherein the target glycanor glycans comprise high mannose N-glycans, bisected and SialylLewis^(X) N-glycans, and/or N-acetyl lactosamine containing N-glycans.

141. The method of any of embodiments 121-140, wherein the target glycanor glycans comprise a fucosylated biantennary complex glycan having noreducing end terminal galactose residues, a fucosylated biantennarycomplex glycan having one reducing end terminal galactose residue, afucosylated biantennary complex glycan having two reducing end terminalgalactose residues, a biantennary complex glycan having no reducing endterminal galactose residues, a biantennary complex glycan having onereducing end terminal galactose residue, a biantennary complex glycanhaving two reducing end terminal galactose residues, a fucosylatedbiantennary complex glycan having two galactose residues and oneN-acetylneuraminic acid residue, a fucosylated biantennary complexglycan having two galactose residues and two N-acetylneuraminic acidresidues, a biantennary complex glycan having two galactose residues andtwo N-acetylneuraminic acid residues, a high mannose glycan having fivemannose residues, a high mannose glycan having six mannose residues, ahigh mannose glycan having seven mannose residues, a high mannose glycanhaving eight mannose residues, and/or a high mannose glycan having ninemannose residues.

142. The method of any of embodiments 121-141, wherein the target glycanor glycans comprise the glycans present in Table E1 or a subset thereof143. The method of any of embodiments 121-142, wherein the target glycanor glycans comprise the glycans detectable in the cell surface glycanprofile.

144. A method for screening one or more test agents or conditions on acell composition, comprising:

(a) assessing a cell surface glycan profile in a sample from a test cellcomposition, wherein the test cell composition is or is derived from asource composition that has been incubated or treated in the presence ofone or more test agents or conditions; and

(b) comparing the cell surface glycan profile of the sample to the cellsurface glycan profile of a reference sample, the reference samplecomprising one or more target glycans.

145. A method for screening one or more test agents or conditions on acell composition, comprising comparing the cell surface glycan profileof a sample compared to the cell surface glycan profile of a referencesample, wherein the sample is from a test cell composition that is or isderived from a source composition that has been incubated or treated inthe presence of one or more test agents or conditions.

146. The method of embodiment 144 or embodiment 145, wherein the cellsurface glycan profile comprises the presence, absence, identity and/orlevel of one or more glycans in the sample.

147. The method of any of embodiments 145-146, wherein the cell surfaceglycan profile is determined according to the method of any ofembodiments 1-93.

148. The method of any of embodiments 144-147, wherein the referencesample is derived from a composition incubated or treated under the sameor substantially the same conditions as the test cell composition orsource composition except in the absence of treating in the presence ofthe one or more test agents or conditions or in the presence of one ormore alternative test agents or conditions.

149. The method of any of embodiments 144-147, wherein the referencesample comprises an average or median of the presence, absence, identityand/or level of the one or more target glycan or glycans among aplurality of compositions incubated or treated in the presence of theone or more test agents or conditions.

150. The method of any of embodiments 144-149, wherein the one or moretest agents or conditions comprises presence or concentration of serum;time in culture; presence or amount of a stimulating agent; the type orextent of a stimulating agent; presence or amount of amino acids;temperature; the source or cell types of the source composition; theratio or percentage of cell types in the source composition, optionallythe CD4+/CD8+ T cell ratio; the presence or amount of beads; celldensity; static culture; rocking culture; perfusion; the type of viralvector; the vector copy number; the presence of a transduction adjuvant;cell density of the source composition in cryopreservation; the extentof expression of the recombinant receptor; or the presence of a compoundto modulate cell phenotype.

151. The method of any of embodiments 144-150, wherein the one or moretest agents or conditions comprises one or more compounds from a libraryof test compounds.

152. The method of any of embodiments 144-151, wherein the targetglycans comprise at least 25, at least 50, at least 60, at least 70, atleast 80, at least 90, at least 100, or at least 200 different speciesof glycans, optionally wherein the species of glycans are N-glycans.

153. The method of any of embodiments 144-152, wherein the target glycanor glycans comprise high mannose N-glycans, bisected and Sialyl Lewis'N-glycans, and/or N-acetyl lactosamine containing N-glycans.

154. The method of any of embodiments 144-153, wherein the target glycanor glycans comprise a fucosylated biantennary complex glycan having noreducing end terminal galactose residues, a fucosylated biantennarycomplex glycan having one reducing end terminal galactose residue, afucosylated biantennary complex glycan having two reducing end terminalgalactose residues, a biantennary complex glycan having no reducing endterminal galactose residues, a biantennary complex glycan having onereducing end terminal galactose residue, a biantennary complex glycanhaving two reducing end terminal galactose residues, a fucosylatedbiantennary complex glycan having two galactose residues and oneN-acetylneuraminic acid residue, a fucosylated biantennary complexglycan having two galactose residues and two N-acetylneuraminic acidresidues, a biantennary complex glycan having two galactose residues andtwo N-acetylneuraminic acid residues, a high mannose glycan having fivemannose residues, a high mannose glycan having six mannose residues, ahigh mannose glycan having seven mannose residues, a high mannose glycanhaving eight mannose residues, and/or a high mannose glycan having ninemannose residues.

155. The method of any of embodiments 144-154, wherein the target glycanor glycans comprise the glycans present in Table E1 or a subset thereof156. The method of any of embodiments 144-155, wherein the target glycanor glycans comprise the glycans detectable in the cell surface glycanprofile.

157. The method of any of embodiments 144-156, comprising selecting theone or more test agent or conditions for incubating or treating thecells if the comparison indicates the cell surface glycan profile of thesample or each of the one or more target glycans is substantially thesame as the reference sample and/or if the comparison indicates the cellsurface glycan profile comprising the one or more target glycans or eachof the one or more target glycans differs by no more than or about 20%,no more than or about 15%, no more than or about 10% or no more than orabout 5% from the cell surface glycan profile or each of the one or moretarget glycans to the total glycans present in the reference sample.

158. The method of any of embodiments 144-156, comprising repeating themethod with one or more further test agent or condition if thecomparison indicates the cell surface glycan profile of the sample oreach of the one or more target glycans is substantially different fromthe reference sample and/or if the comparison indicate the cell surfaceglycan profile comprising the one or more target glycans or each of theone or more target glycans differs by greater than or greater than about10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more from the cellsurface glycan profile or each of the one or more target glycans to thetotal glycans present in the reference sample.

159. The method of any of embodiments 144-156, wherein the test agent isa candidate for modulating the growth, proliferation, viability,differentiation, activation and/or expansion, of one or more cells inthe test cell composition.

160. The method of any of embodiments 144-159, wherein the test cellcomposition comprises cells comprising a recombinant nucleic acid.

161. The method of any of embodiments 125-160, wherein the recombinantnucleic acid encodes a recombinant protein, optionally a recombinantreceptor.

162. The method of embodiment 160, wherein the recombinant receptor isor comprises a chimeric receptor and/or a recombinant antigen receptor.

163. The method of embodiment 161 or embodiment 162, wherein therecombinant receptor is capable of binding to a target antigen that isassociated with, specific to, and/or expressed on a cell or tissue of adisease, disorder or condition.

164. The method of embodiment 163, wherein the disease, disorder orcondition is an infectious disease or disorder, an autoimmune disease,an inflammatory disease, or a tumor or a cancer.

165. The method of embodiment 163 or embodiment 164, wherein the targetantigen is a tumor antigen.

166. The method of any of embodiments 163-165, wherein the targetantigen is selected from among ROR1, B cell maturation antigen (BCMA),carbonic anhydrase 9 (CAIX), tEGFR, Her2/neu (receptor tyrosine kinaseerbB2), L1-CAM, CD19, CD20, CD22, mesothelin, CEA, and hepatitis Bsurface antigen, anti-folate receptor, CD23, CD24, CD30, CD33, CD38,CD44, EGFR, epithelial glycoprotein 2 (EPG-2), epithelial glycoprotein40 (EPG-40), EPHa2, erb-B2, erb-B3, erb-B4, erbB dimers, EGFR vIII,folate binding protein (FBP), FCRLS, FCRHS, fetal acetylcholinereceptor, GD2, GD3, HMW-MAA, IL-22R-alpha, IL-13R-alpha2, kinase insertdomain receptor (kdr), kappa light chain, Lewis Y, L1-cell adhesionmolecule, (L1-CAM), Melanoma-associated antigen (MAGE)-A1, MAGE-A3,MAGE-A6, Preferentially expressed antigen of melanoma (PRAME), survivin,TAG72, B7-H6, IL-13 receptor alpha 2 (IL-13Rα2), CA9, GD3, HMW-MAA,CD171, G250/CAIX, HLA-AI MAGE A1, HLA-A2 NY-ESO-1, PSCA, folatereceptor-a, CD44v6, CD44v7/8, avb6 integrin, 8H9, NCAM, VEGF receptors,5T4, Foetal AchR, NKG2D ligands, CD44v6, dual antigen, a cancer-testesantigen, mesothelin, murine CMV, mucin 1 (MUC1), MUC16, PSCA, NKG2D,NY-ESO-1, MART-1, gp100, oncofetal antigen, ROR1, TAG72, VEGF-R2,carcinoembryonic antigen (CEA), Her2/neu, estrogen receptor,progesterone receptor, ephrinB2, CD123, c-Met, GD-2, O-acetylated GD2(OGD2), CE7, Wilms Tumor 1 (WT-1), a cyclin, cyclin A2, CCL-1, CD138, apathogen-specific antigen and an antigen associated with a universaltag.

167. The method of any of embodiments 163-166, wherein the recombinantreceptor is or comprises a functional non-TCR antigen receptor or a TCRor antigen-binding fragment thereof.

168. The method of any of embodiments 163-167, wherein the recombinantreceptor is a chimeric antigen receptor (CAR).

169. The method of any of embodiments 121-168, wherein the cellscomprise mammalian cells or the test cell composition comprisesmammalian cells.

170. The method of any one of embodiments 121-169, wherein the cellscomprise human cells or the test cell composition comprises human cells.

171. The method of any one of embodiments 121-168, wherein the cellscomprise stem cells or the test cell composition comprises stem cells.

172. The method of embodiment 171, wherein the stem cell is an inducedpluripotent stem cell (iPSC).

173. The method of any of embodiments 121-170, wherein the compositionor test cell composition comprises cells present in an apheresis productor a leukapheresis product or cells derived therefrom.

174. The method of any of embodiments 121-170 and 1713, wherein:

the cells comprise immune cells, white blood cells, peripheral bloodmononuclear cells (PBMC), lymphocytes, or unfractionated T cells; or

the test cell composition comprises immune cells, white blood cells,peripheral blood mononuclear cells (PBMC), lymphocytes, orunfractionated T cells.

175. The method of any one of embodiments 121-174, wherein the cellscomprise an immune cell or the test cell composition comprises immunecells.

176. The method of embodiment 175, wherein the immune cell is a T cell,B cell, macrophage, neutrophil, natural killer (NK) cell or dendriticcell.

177. The method of any of embodiments 121-176, wherein the cellscomprise T cells that are CD4+ and/or CD8+ T cells or the test cellcomposition comprises T cells that are CD4+ and/or CD8+ T cells.

178. The method of any of embodiments 1-177, wherein the cells areprimary cells.

179. A method of detecting a presence, absence, identity, and/or levelof one or more substances in a cell composition, the method comprising:

(a) assessing the cell surface glycan profile in a sample from a testcell composition comprising a plurality of cells according to the methodof any of embodiments 1-93, wherein the plurality of cells are from orare derived from a cell type; and

(b) identifying one or more non-native glycans in the cell surfaceglycan profile that are not synthesized and/or expressed by cells of thecell type.

180. The method of embodiment 179, wherein the cell type is human.

181. The method of embodiment 179 or 180, wherein the cell type is animmune cell.

182. the method of any of embodiments 180 or 181, wherein the cell typeis a T cell.

183. The method of any of embodiments 179-182, wherein the test cellcomposition was produced by culturing and/or incubating the plurality ofcells in the presence of the substance, said substance comprising atleast one protein comprising one or more non-native glycans.

184. The method of embodiment 183, wherein the at least one protein isan albumin, a growth factor, a cytokine, a chemokine, an insulin orinsulin-like peptide, a transferrin, or a superoxide dismutase.

185. The method of embodiment 183 or 184, wherein the at least oneprotein is a recombinant protein.

186. The method of any of embodiments 183-185, wherein the one or morenon-native glycans and/or the at least one protein are present in aserum.

187. The method of embodiment 186, wherein the serum is fetal bovineserum (FBS), bovine calf serum (BCS), newborn calf serum (NBCS), horseserum, goat serum, lamb serum, donkey serum, or porcine serum.

188. The method of any of embodiments 179-187, wherein the one or morenon-native glycans and/or the at least one protein are (i) not producedby and/or (ii) not expressed on the surface of cells from the sameorder, family, genus, or species as the cells of the plurality.

189. The method of any of embodiments 179-188, wherein the one or morenon-native glycans comprise a non-human glycan.

190. The method of any of embodiments 179-189, wherein the identifyingthe one or more non-native glycans comprises comparing the surfaceglycan profile to a reference glycan profile, wherein the referencesample is a glycan profile from a source containing the one or morenon-native glycans.

191. The method of embodiment 189 or 190, wherein the reference glycanprofile is generated from a reference sample comprising a substance thathas not been contacted, incubated, and/or exposed to the cells of theplurality, optionally wherein the substance is or comprises a media, aserum, or a component thereof, or is a protein or recombinant proteinthereof.

VI. EXAMPLES

The following examples are included for illustrative purposes only andare not intended to limit the scope of the invention.

Example 1: Mapping Surface N-linked Glycans of a Cell Composition

To map the cell surface N-linked glycan profile of a cell composition,exemplary cryopreserved T cell compositions containing cells expressinga chimeric antigen receptor (CAR) were individually thawed, diluted 1:10in cell culture media heated to 37° C., and a sample of 1-2.5×10⁶ cellswas transferred to a fresh tube.

The transferred cell sample was centrifuged and washed in phosphatebuffered saline (PBS), followed by reconstitution of the cell suspensionwith approximately 198 μl PBS. Approximately 2 μL PNGase F PRIME (N-ZymeScientifics; available from Bulldog Bio, Catalog No. NZPP050) was addedto the cell suspension containing whole, intact cells, followed byincubation for 30 minutes at 37° C. with gentle mixing. To obtainreleased surface N-glycans, the cell suspension was centrifuged and thesupernatant was collected into a clean tube that was immediatelyevaporated to dryness with vacuum centrifugation.

The dried down N-glycans were reconstituted in 30 μl water and 5 μL of alabeling reagent composed of an N-hydroxysuccinimide (NHS) carbamatetagging group, a quinolone fluorophore, and a basic tertiary amine(Glycoworks™ RapiFluor-MS™ label, Catalog No. 715004793, WatersCorporation, MA) was added. The sample was mixed, incubated forapproximately 5 minutes at room temperature, following by quenching ofthe label by adding approximately 365 μl acetonitrile to the sample. Thesample was mixed and centrifuged. Solid phase extraction (SPE) wascarried out on the sample to further clean up the N-glycans prior tofurther analysis. The N-glycan sample was evaporated to dryness with avacuum centrifuge and then resuspended in appropriate diluent foranalysis.

Glycans were separated using HILIC liquid chromatography and detected byfluorescence (Waters ACQUITY I-Class) (HILIC-FLR) and mass spectrometry(positive electrospray ionization (ESI), Q-Exactive™ HF (ThermoScientific)), i.e., HILIC-ESI-MS, for relative quantification andidentification.

Table E1 set forth the retention time(s) (RT), as well as themonoisotopic mass and the mass of the +2, +3, and/or +4 charge states,for exemplary sugars detected using this method.

TABLE E1 Retention Time and Mass of Exemplary Glycans Sugar RT +2 +3 +4Mass A2G0F 13.15 887.8699 1773.74 A1G1F   14, 14.5 867.36 1732.72 M514.35 773.81 1545.62 A2G1 14.88 895.8674 1789.735 A2G1F 15.4&15.9 968.91935.8 A1S1 16.1 939.8756 1877.751 A3G1F 16.4, 17.1 1070.437 2138.873A4G0F 1090.95 2179.9 A1S1F 16.5 1012.905 2023.81 m6 16.9 854.84071707.681 A2G1S1 16.9, 17.4 1041.417 2080.833 M5A1G0F/M4A1G1F 16.9948.3837 1894.767 m6 17.1 854 1706 A2G2 17.2 976.89 1951.78 A2G1S1F17.6, 18.3 1114.445 2226.889 M4A1S1 18.3? 1020.9 2039.8 A3G2 17.91078.43 2154.86 A2G2F 18.14 1049.92 2097.84 M5A1G1 18.5 956.38111910.762 A3G2F 18.7, 19.6 1151.464 2300.928 M5A1G1F 18.85, 19.9 1029.411 2056.821 M4A1S1F 20.6 1093.933 2185.865 A2G2S1 19.3-19.5&20.751122.443 2242.886 M7 19.7 935.8676 1869.735 A2G2S1F 20.1, 20.3, 21.51195.473 2388.945 M5A1S1 20.6, 21.9 1101.93 2201.861 A2G2S1F 21.2-21.61195.47 2388.94 M5A1S1F 22 1174.96 2347.92 A2G2S2 21.6, 22.7 1267.9942533.987 M8 22.1 1016.895 2031.789 A3G2S1F(bisected) 1297.01 2592.021039.884 2077.769 A2G2S2F 22.3 1341.021 2680.042 M5A1S1F 22.8 1174.962347.92 A2G2S2F 23.35 1340.979 2679.959 A2S1F2 23.45 1268.5 2535 A3G3S123.6 1305.01 2608.02 A2S2 23.7 1267.994 2533.987 M9 23.95 1097.9222193.844 A3G3S1F 24 1378.04 2754.08 A2G2S2F 24.4 1341.021 2680.042A3G3S1F 24.6 1378.042 2754.083 A3G2S2F(bisected) 25.02 1442.56 962.042883.12 A2S2F2 25.25 1414.05 2826.1 a3g3s2 1450.56 2899.12 M9 + hex25.65 1178.949 2355.897 A3G3S2F 25.7, 26.3, 26.6 1016.061 3045.183A4G4S1F(Or A3 w/LacNac) 26.7 1040.74 3119.221 A3G3S1F 26.7 1378.042754.08 A3S3 27.65 1064.407 3190.221 A4G4S2F(or A3 w/LacNac) 27.951137.772 3410.315 A3S3F2 1161.78 3482.34 A3S3F 27.2, 28.1 1113.0933336.28 A4G4S2F2(or A3 w/LacNac) 1186.46 3556.38 A4G4S3F(or A3 w LacNac)29.22 1234.804 3701.411 A3S4F 29.5 1210.126 3627.379 A3S4F2 30 1258.8123773.435 A4S4F 31 1331.84 3992.52 A4G4S3F w 1lacnac(or A3 w 31 1356.524066.56 2LacNac) A4G4S2F w 2lacnac 31 1381.2 4140.6 A4G4S5F 1428.874283.61 A4G4S4F w 1lacnac 32 1453.55 4357.65 A4G4S3F w 2lacnac(or A3 w33 1478.23 4431.69 3LacNac) a4g4s2f w 3lacnac 34 1502.91 4505.73 A4G4S4Fw 2lacnac 34 1575.27 4722.81 A4G4S3F w 3lacnac 35 1599.95 4796.85A4G4S5F + 2lacnac 1672.3 5013.9 A4G4S4 w 3lacnac 35.3 1696.98 5087.94A4G4S3F w 4lacnac 1721.65 1291.49 5161.959 1527.25 1145.939 4579.757A4G4S4F w 4lacnac 36 1364.264 5453.058 A4G4S4F w 3lacnac 1272.985087.919 1291.49 5161.959 A4G4S5F W 3lacnac 1345.754 5379.015 a4G4S3F W5lacnac 1382.773 5527.091 A4G4S2F w 6lacnac 1401.281 5601.125 A4G4S4F w5lacnac 1455.547 5818.189 A4G4S5F w 4lacnac 1437.039 5744.154 A4G4S2F w7lacnac 1492.562 5966.247 1419.536 5674.143 A4G4S4F w 6lacnac 1546.836183.321 A4G4S3F w 7lacnac 1565.34 6257.359 A4G4S4F w 7lacnac 401638.113 6548.452 A4G4S3F w 8lacnac 40 1656.626 6622.506 A4G4S4f w 8lacnac 1729.404 6913.617 A4G4S3F w 9lacnac 1747.902 6987.607

The method described above was carried out on an exemplary T cellcomposition generated by a process involving separately generating CD4+and CD8+ populations of engineered T cells derived from the same subjectfollowing immunoaffinity-based enrichment of the CD4+ and CD8+ cellpopulations from a leukapheresis sample from a healthy human donor.Isolated CD4+ and CD8+ T cell populations were activated and transducedwith a viral vector encoding an anti-CD19 CAR, followed by expansion andcryopreservation of the engineered cell populations. The anti-CD19 CARcontained an anti-CD19 scFv derived from a murine antibody, anIg-derived spacer, a human CD28-derived transmembrane domain, a human4-1BB-derived intracellular signaling domain and a human CD3zeta-derived signaling domain. An annotated HILIC-FLR chromatogram ofcell surface N-glycans from the engineered CD8+ T cell composition isdepicted in FIG. 2A, and demonstrated a complex mixture of N-glycantypes, including the presence of high mannose N-glycans, bisectedglycans, and sialyl Lewis' glycans, were present on the surface of cellsin the composition.

In another example, the method was carried out on an alternativeexemplary anti-CD19 CART cell composition generated by a processinvolving immunoaffinity-based selection of T cells (including CD4+ andCD8+ cells) from a leukapheresis sample, followed by activation andtransduction with a viral vector encoding the anti-CD19 CAR, expansionand cryopreservation. The CAR contained an anti-CD19 scFv derived from amurine antibody, a region of CD28 including an extracellular region, atransmembrane domain and a costimulatory region, and a CD3-zetaintracellular signaling domain. The annotated HILIC-FLR chromatogram inFIG. 3A and total ion chromatogram (TIC) in FIG. 3B, produced by theHILIC-ESI-MS analysis of the composition, similarly demonstrated anN-glycan profile containing a complex mixture of N-glycan types.

These results confirmed that it was possible to obtain high resolutionmaps of N-glycans released from the surface of whole, intact cells,thereby demonstrating that the method can be used to generate anN-glycan map of cell compositions to characterize the whole cell surfaceglycosylation state.

Example 2: Identification of Glycan Species by HILIC and Tandem PositiveElectrospray Ionization Mass Spectrometry (ESI-MS/MS)

To confirm identification of glycans, high resolution mass spectrometrywas employed to identify glycans, including isobaric species resolved byHILIC. The analysis was carried out on an exemplary compositioncontaining activated CD3+ T cells that were activated followingstimulation with anti-CD3/anti-CD28, e.g., coated on magnetic beads.N-glycans were extracted, labeled, and processed as described inExample 1. HILIC-ESI-MS was performed substantially as described inExample 1, except using tandem mass spectrometry (MS/MS). N-Glycans wereseparated by HILIC and detected by fluorescence (HILIC-FLR) andESI-MS/MS provided relative quantification and identification.

As shown in FIG. 4A, the annotated HILIC-FLR chromatogram revealed thatthe cell surface N-glycan map of anti-CD3/anti-CD28 activated CD3+ cellswas a complex mixture of glycan types.

For tandem mass spectrometry (MS/MS), particles were ionized by ESI andseparated by their mass-to-charge ratio at the first stage of the massspectrometry. FIG. 4B depicts an extracted ion chromatogram (XIC)produced from the first stage of mass spectrometry for the exemplaryA3S3F N-glycan in the +3 charged state using a 5 ppm mass tolerance.Multiple chromatographic peaks of the N-glycan with identical isotopicdistributions were observed, which is consistent with the likelypresence of linkage differences between the monosaccharide units of theglycan structure. After the first stage, the glycans underwentfragmentation and the resulting fragments were separated and measured inthe second stage of the MS/MS. The MS/MS fragmentation of the exemplaryN-glycan A3S4F, fragmented by high energy collisional dissociation (HCD)at a normalized collision energy (NCE) of 15, is shown in FIG. 4C.Combined with the high resolution MS data of the first stage, thefragmentation of A3S4F confirmed the presence of an n-acteyl neuraminicacid linkage at the canonical terminal galactose residue, as well as ata unique site on an n-acetyl glucosamine residue of the same antennae(FIG. 4C, boxes). These results demonstrate that MS/MS combined with thehigh resolution MS data can identify glycan structures, including uniqueglycan structures, following separation and detection by HILIC-FLR.

Example 3: Comparison of N-linked Glycan Profiles of Different CellCompositions

The N-glycan mapping profile of whole, intact cells described in Example1 was used to compare the N-glycan profile among different cellcompositions.

A. Impact of Process Features for Engineering CD4+ and CD8+ CAR+ T cellcompositions on N-Glycan Profiles

Cell compositions obtained during various steps of an exemplary processof producing T cells engineered with a chimeric antigen receptor (CAR)were compared to investigate the impact of the process on T cell surfaceN-glycans in the cell product. The exemplary process generally involvedisolation of bulk PBMCs followed by immunoaffinity-based selectionresulting in selected CD4+ T cells and selected CD8+ T cells, which werethen cryopreserved. The thawed, selected CD4+ and/or CD8+ T cells wereseparately incubated with a bead-based activation reagent and transducedwith a lentiviral vector encoding the CAR. The transduced cells wereincubated and expanded in the presence of cytokines and subsequentlycryopreserved. Specifically, the following compositions were analyzed:(1) cell composition containing bulk PBMCs, (2) thawed cryopreservedcell compositions containing selected CD4+ or CD8+ T cells selected byimmunoaffinity-based selection; (3) cell compositions containingselected T cells incubated with anti-CD3/anti-CD28 beads in the presenceof IL-2, IL-15 and/or IL-7 for 18-24 hours at 37° C.; (4) cellcompositions that were transduced with a lentiviral vector encoding theCAR; and (5) thawed, cryopreserved cell compositions containingCAR-expressing cells that were subjected to expansion in the presence ofIL-2, IL-15 and/or IL-17 cytokines prior to cryopreservation. TheN-glycan profile of whole, intact cells from the each of the above cellcompositions was assessed using the procedures substantially asdescribed in Example 1.

Bulk PBMCs exhibited a highly heterogeneous population of N-glycansincluding bi-, tri- and tetra-antennary hybrid and complex type sugars.Selection of T cells (CD4+ and CD8+) reduced the heterogeneity of theglycan pattern, resulting in primarily bi- and tri-antennary sialyatedglycans with an increase in tetra-antennary species compared to the bulkcell population. CD4+ cells and CD8+ cells activated byanti-CD3/anti-CD28 co-stimulation, retained the bi- and tri-antennaryspecies, while tetra-antennary species were reduced.

Comparison of the N-glycan profiles among T cell compositions containingselected CD4+ or CD8+ T cell compositions, CAR-transduced cells orCAR-transduced cells that were further expanded and cryopreserved, thesame mixture of N-glycans, including high mannose N-glycans, bisectedglycans, and sialyl Lewis^(X) glycans, were present in each of the Tcell compositions but at different levels. FIGS. 5A-5C provide overlaysof cell surface N-glycan maps comparing levels of specific high mannoseN-glycans (FIG. 5A), bisected and sialyl Lewis^(X) N-glycans (FIG. 5B),and N-acetyl lactosamine containing N-glycans (FIG. 5C) in CD4+ and CD8+cells among the cell compositions. These results are summarized in TableE2, which sets forth the percentage of N-glycans that were high mannoseN-glycans, bisected and sialyl Lewis^(X) N-glycans, and N-acetyllactosamine containing N-glycans in each of the CD4+ and CD8+compositions.

The results generally showed increased surface high mannose speciesglycans, increased Sialyl Lewis^(X)N-glycans and decreased surfacepolylactosamine containing N-glycans in CD4+ and CD8+ cells fromcompositions containing CAR-expressing cells that were further expandedand cryopreserved compared to the selected CD4+ and CD8+ T cellcompositions or CAR-transduced cell compositions. Without wishing to bebound by theory, it is hypothesized that in some cases these results maybe due to changes in N-glycans occurring in the cell composition duringexpansion and/or activation. Within cell product compositions that weresubjected to further expansion and cryopreservation, differences alsowere observed between CD4+ and CD8+ cell compositions, with CD8+ cellcompositions exhibiting substantially more bisected N-glycans on theirsurface relative to the CD4+ cell compositions.

TABLE E2 Percentage of N-glycans in CD4+ and CD8+ cell compositions %N-acetyl % Bisected/Sialyl lactosamine Cell % High Mannose Lewis^(X)containing Composition CD4 CD8 CD4 CD8 CD4 CD8 Selected T 9.3 10.5 1.71.8 33.3 22.5 cells Transduced T 10 10.1 2.8 3.6 17 16.6 cellsTransduced 17.4 17.1 5.7 9.9 13 13.8 and expanded T cells

B. Impact of Process Features on N-Glycan Profile of T Cell Compositions

Cell compositions employed during engineering of CAR+ T cells wereassessed for impact of process features, e.g. selection and activation,on N-glycan profiles. CD3+ T cells were selected from bulk PBMCs byimmunoaffinity-based selection and cryopreserved. Following thaw, theselected CD3+ T cells were incubated with an anti-CD3/anti-CD28bead-based activation. Specifically, the following compositions wereanalyzed: (1) cell composition containing peripheral blood mononuclearcells, (2) cell composition containing selected CD3+ T cells selected byimmunoaffinity-based selection; and (3) cell composition containingselected T cells incubated with anti-CD3/anti-CD28 beads. The N-glycanprofile of whole, intact cells from the each of the above cellcompositions was assessed using the procedures substantially asdescribed in Example 1.

As shown in the exemplary HILIC-FLR chromatograms in FIG. 6, differenceswere observed in the relative levels of surface N-glycans, particularlyfor expressed biantennary/hybrid, bisected core, and N-acetyllactosamine repeat N-glycans. Exemplary areas of changes are highlightedin FIG. 6 based on the degree of increase (+ or ++) or decrease (−)between one or more of the other compositions. These results areconsistent with a conclusion that the steps for processing cells togenerate engineered T cells can influence surface N-glycan profiles.

C. N-Glycan Profile of Different CAR-T Cell Compositions

The surface N-linked glycan profiles of CD4+ and CD8+ T cellcompositions comprising T cells expressing an anti-CD19 CAR describedabove were compared to the N-glycan profile of a cell compositioncontaining T cells expressing an alternative anti-CD19 CAR and a cellcomposition containing T cells expressing a CAR that recognizes analternative target antigen. The alternative anti-CD19 CAR contained ananti-CD19 scFv derived from a murine antibody (which was different fromthe anti-CD19 scFv of the anti-CD19 CAR described above), a region ofhuman CD28 including an extracellular region, a transmembrane domain anda costimulatory region, and a human CD3-zeta intracellular signalingdomain. The CAR that recognizes an alternative target antigen compriseda human scFv, a spacer region, a CD28 transmembrane domain, a4-1BB-derived intracellular co-signaling sequence, and a CD3-zetaderived intracellular signaling domain. The process to produce each ofthe cell compositions also differed with respect to various features,such as the presence, number or ratio of CD4+ and CD8+ cells, and/or theconditions for activation, transduction and/or expansion.

The N-glycan profile of whole, intact cells from the cell compositionswere assessed using the procedures substantially as described inExample 1. As shown in FIG. 7, each of the analyzed compositionsexhibited a unique cell surface N-glycan profile. The percentage ofexemplary types of N-glycans in each cell composition are summarized inTable E2. In some cases, N-acetylactosamine exists as repeats present ina polylactosamine glycan.

In the composition containing the alternative anti-CD19 CAR,approximately 1% of cell surface N-glycans were bisected N-glycans. Thisresult was consistent across more than twelve tested compositionscomprising cells expressing the alternative anti-CD19 CAR produced usinga similar process. The N-glycan profile from the cell compositioncontaining cells expressing the CAR that recognizes an alternativetarget antigen demonstrated high levels of bisected N-linked glycans andSialyl Lewis' N-glycans.

TABLE E3 Percentage of exemplary types of N-glycans AlternativeAnti-CD19 Anti-CD19 anti-CD19 CAR CAR CD4+ CAR CD8+ Alternative T cell Tcell T cell CAR T cell composition composition composition compositionPurpose/Role N-acetyl 14.2 13 13.8 11.5 Galectin* Binding lactosamineHigh 11.9 17.4 17.1 14.3 Immature/ Mannose Incomplete ProcessingBisected 0.9 2.8 6.9 11.3 Reduces Galectin Binding Sialyl Lewis 3 2.9 36 Selectin* Binding

D. Analysis of N-Glycan Profiles of Engineered Cells Generated byDifferent Processes

The surface N-linked glycan profiles of three different CD4+ T cellcompositions comprising T cells expressing the exemplary anti-CD19 CARdescribed above in Example 3A were obtained by substantially the samemethods as described in Example 1. Two of the CD4+ T cell compositionswere obtained from a sample from the same individual subject, one ofwhich was produced by a method similar to the exemplary engineeringprocess described in Example 3A (“exemplary process”) and the other CD4+T cell composition was produced by an exemplary alternative engineeringprocess (“alternative process”). A second CD4+ T cell composition froman additional subject sample was produced from the alternative method.

The N-glycan profile of whole, intact cells from the cell compositionswere assessed using the procedures essentially as described inExample 1. As shown in FIG. 8, differences were observed amongindividual peaks of the surface N-glycan profiles. The observed bisectedand Sialyl-Lewis^(X) N-glycan content obtained from the CD4+ T cellcompositions are summarized in Table E4.

TABLE E4 N-glycan content in CD4+ T cell compositions containinganti-CD19 CAR expressing cells Engineering % Bisected % Sialyl-LewisSubject Process N-glycans N-glycans 1 Alternative 9.8 3.4 1 Exemplary1.9 2.0 2 Alternative 1.6 1.6

To further assess if different variables used during a cell engineeringprocess may result in different surface N-glycan profiles, three CD4+and three CD8+ T cell compositions containing T cells expressing theexemplary anti-CD19 CAR were generated from samples obtained from thesame individual subject. The T cell compositions were essentiallygenerated by the substantially the same engineering process, with theexception that they differed in aspects related to two conditions usedin the process for engineering and cultivating the cells. As shown inFIGS. 9 and 10, the surface N-glycan profiles observed in the CD4+(FIG.9) and the CD8+(FIG. 10) T cell compositions appeared consistent,although changes in specific peaks corresponding to bisected glycans(arrows) were observed.

E. N-Glycan Surface Profiles of T Cell Compositions Containing CARExpressing Cells

The surface N-linked glycan profiles of T cell compositions comprising Tcells expressing either the exemplary anti-CD19 CAR described above inExample 3A or a CAR with an alternative target antigen were obtained bythe method described in Example 1. Two compositions were generated fromsamples obtained from the healthy subjects, and one was obtained from asubject having a disease associated with the target antigen (patient).

The N-glycan profiles of whole, intact cells from the cell compositionswere assessed using the procedures essentially as described inExample 1. As shown in FIGS. 11 and 12, differences were observed amongindividual peaks of the surface N-glycan profiles (see arrows) obtainedfrom the anti-CD19 CAR CD8+ T cell compositions (FIG. 11) and thealternative CAR T cell compositions (FIG. 12). Differences betweenbisected and Sialyl-Lewis^(X) N-glycan content were also observed andare summarized in Table E5 and E6.

TABLE E5 N-glycan content in CD8+ T cell compositions containinganti-CD19 CAR expressing cells % Bisected % Sialyl-Lewis Subject StatusN-glycans N-glycans 1 Healthy 19.0 0.9 2 Healthy 8.4 4.9 3 Patient 6.56.7

TABLE E6 N-glycan content in T cell compositions containing cellsexpressing a CAR that targets an alternative antigen % Bisected %Sialyl-Lewis Subject Status N-glycans N-glycans 1 Healthy 6.4 6.3 2Patient 8.6 6.5 3 Healthy 7.7 7.5

The results show that different degrees of variability of surface Nglycan profiles may exist among different CAR T cell compositionsgenerated from different donors. This observation is consistent with autility of surface N-glycan profiling to monitor changes inglycosylation during a process for generating cells and to utilizeN-glycan profiles as a parameter to monitor uniformity of cellcompositions generated from different donors and/or using the same ordifferent processes.

Example 4: Detection of Substances with Surface N-glycan Profiling

The ability of surface N-glycan profiling to detect glycans originatingfrom various substances (for example, potential residual substances,such as residual proteins present in cell compositions) was tested. Suchsubstances may include serum proteins, cytokines or growth factors usedin a process for engineering cells, including cells expressingrecombinant receptors, e.g. CAR.

As an example of a substance, a sample of fetal bovine serum (FBS) wasincubated with PNGase F PRIME. N-glycans were collected from the sampleand dried down. The N-glycans were then reconstituted in water andlabeled, cleaned by SPE, and analyzed with HILIC-ESI-MS similar to asdescribed in Example 1. As shown in FIG. 13 (top panel) a glycan profilewas generated, consistent with an ability of glycan profile mapping todetect N-glycans bound to FBS.

N-glycan profiles were generated from CD4+ and CD8+ T cell compositionscontaining T cells expressing the exemplary anti-CD19 CAR described inExample 3A. For each sample from an individual subject, two CART cellcompositions were generated by different processes that differed in theconditions used for activating and cultivating the cells. Both processesinvolved incubations with media containing FBS. Surface N-glycanprofiles were generated as described in Example 1.

Exemplary surface N-glycan profiles from CD4+ CAR T cell compositionsproduced by the different processes are shown in FIG. 13. Glycansoriginating from FBS were detected in all of the examined CAR T cellcompositions (arrows). The detected N-glycans originating from FBSincluded an alpha-gal (AG) and an NGNA epitope. These epitopes are notpresent in primates, consistent with a non-human source, e.g., FBS. Thealpha-gal epitope made up less than 1% of the total N-glycan profilesmeasured from both cell compositions. In this experiment, the FBSN-glycans were observed in profiles from the CAR T cell compositions inthe same proportions as observed from the FBS sample, consistent withFBS as the source of the glycans. Differences in residual FBS N-glycansdetected in N-glycan profiles were observed in CAR T cell compositionsgenerated by the different processes. No non-human glycans were observedin a cell composition that was generated by a process that employedserum-free media.

Example 5: Effects of Surface N-Glycan Levels on T Cell Activity

Mixed CD4+ and CD8+ T cell compositions containing cells expressing aCAR were generated from a healthy individual subject and cryofrozen. TheCAR T cell compositions were thawed, rinsed, and then were treated for30 minutes with PNGase F (PNGase F Prime) to remove surface glycans fromthe cells or Sialidase A 51 to remove terminal sialic acid residues fromthe cells. As a negative control, cells were incubated DPBS alone.

Cells from the CART cell compositions were rinsed, and 2.5×10⁴ CAR+ Tcells per well were incubated with anti-CD3/anti-CD28 antibodyconjugated beads (bead stimulation) or co-cultured with cells thatendogenously expressed the target antigen of the CAR (antigenstimulation). After 14-18 hours, media was collected from the cellcultures and the extracellular IFN-gamma was quantified with a IFN-gammaAlphaLISA kit.

As shown in Table E7, removal of N-glycans or terminal sialic acidresidues from the cell surface of the CAR T cells resulted in anincreased production of IFN-gamma following stimulation by theanti-CD3/anti-CD28 antibody conjugated beads as compared to the controltreated cells. Removal of surface N-glycans also increased extracellularlevels of IFN-gamma following the co-culture with target antigenexpressing cells. These data are consistent with a role of surfaceN-glycans in impacting T-cell activity.

TABLE E7 Extracellular IFN-gamma levels following stimulation of CAR Tcell compositions Bead Stimulation Antigen stimulation Final [IFNγ] %Difference Final [IFNγ] % Difference (pg/mL) from CNTRL (pg/mL) fromCNTRL Control 4246 24775 N-Glycan removal 5993 34 28229 13 Terminalsialic acid removal 5127 19 25111 1

The present invention is not intended to be limited in scope to theparticular disclosed embodiments, which are provided, for example, toillustrate various aspects of the invention. Various modifications tothe compositions and methods described will become apparent from thedescription and teachings herein. Such variations may be practicedwithout departing from the true scope and spirit of the disclosure andare intended to fall within the scope of the present disclosure.

SEQUENCES SEQ ID NO. SEQUENCE DESCRIPTION 1mrkllifsis aylmagivsc kgvdsatpvt edrlalnavn apadntvnik tfdkvknafgAmino acid dglsqsaegt ftfpadvttv ktikmfikne cpnktcdewd ryanvyvknksequence ofttgewyeigr fitpywvgte klprgleidv tdfksllsgn telkiytetw lakgreysvdPNGase F fromfdivygtpdy kysavvpviq ynkssidgvp ygkahtlglk kniqlptnte kaylrttisgFlavobacterium wghakpydag srgcaewcfr thtiainnan tfqhqlgalg csanpinnqsmeningosepticum pgnwapdrag wcpgmavptridvlnnsltg stfsyeykfq swtnngtngdafyaissfvi aksntpisapvvtn 2 ESKYGPPCPPCP spacer (IgG4hinge) (aa)Homo sapiens 3 GAATCTAAGTACGGACCGCCCTGCCCCCCTTGCCCT spacer(IgG4hinge) (nt) homo sapiens 4ESKYGPPCPPCPGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFY Hinge-CH3 spacerPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQ Homo sapiensEGNVFSCSVMHEALHNHYTQKSLSLSLGK 5ESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVD Hinge-CH2-CH3VSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVL spacerHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPS Homo sapiensQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLS LSLGK 6RWPESPKAQASSVPTAQPQAEGSLAKATTAPATTRNTGRGGEEK IgD-hinge-FcKKEKEKEEQEERETKTPECPSHTQPLGVYLLTPAVQDLWLRDKA Homo sapiensTFTCFVVGSDLKDAHLTWEVAGKVPTGGVEEGLLERHSNGSQSQHSRLTLPRSLWNAGTSVTCTLNHPSLPPQRLMALREPAAQAPVKLSLNLLASSDPPEAASWLLCEVSGFSPPNILLMWLEDQREVNTSGFAPARPPPQPGSTTFWAWSVLRVPAPPSPQPATYTCVVSHEDSRTL LNASRSLEVSYVTDH 7LEGGGEGRGSLLTCGDVEENPGPR T2A artificial 8MLLLVTSLLLCELPHPAFLLIPRKVCNGIGIGEFKDSLSINATNIKHF tEGFRKNCTSISGDLHILPVAFRGDSFTHTPPLDPQELDILKTVKEITGFLLI artificialQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSIATGMVGALLLLLVVALGIGLFM 9 FWVLVVVGGVLACYSLLVTVAFIIFWVCD28 (amino acids 153-179 of Accession No. P10747) Homo sapiens 10IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP CD28 (aminoFWVLVVVGGVLACYSLLVTVAFIIFWV acids 114-179 of Accession No. P10747)Homo sapiens 11 RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYR CD28 (amino Sacids 180-220 of P10747) Homo sapiens 12RSKRSRGGHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYR CD28 (LL to S GG) Homo sapiens13 KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL 4-1BB (aminoacids 214-255 of Q07011.1) Homo sapiens 14RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRG CD3 zetaRDPEMGGKPRRKNPQEGLYN ELQKDKMAEA Homo sapiens YSEIGMKGER RRGKGHDGLYQGLSTATKDTYDALHMQALP PR 15 RVKFSRSAEPPAYQQGQNQLYNELNLGRREEYDVLDKRRGCD3 zeta RDPEMGGKPRRKNPQEGLYN ELQKDKMAEA Homo sapiensYSEIGMKGER RRGKGHDGLY QGLSTATKDTYDALHMQALP PR 16RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRG CD3 zetaRDPEMGGKPRRKNPQEGLYN ELQKDKMAEA Homo sapiens YSEIGMKGER RRGKGHDGLYQGLSTATKDTYDALHMQALP PR 17PGGG-(SGGGG)5-P- wherein P is proline. G is glycine and S is linkerserine 18 GSADDAKKDAAKKDGKS linker 19RKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFR tEGFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAF artificialENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSIATGMVGALLLLLVVALGIGLFM 20 EGRGSLLTCGDVEENPGP T2A 21GSGATNFSLLKQAGDVEENPGP P2A 22 ATNFSLLKQAGDVEENPGP P2A 23QCTNYALLKLAGDVESNPGP E2A 24 VKQTLNFDLLKLAGDVESNPGP F2A 25gacatccagatgacccagaccacctccagcctgagcgccagcctgggcgaccgggtgacca Sequencetcagctgccgggccagccaggacatcagcaagtacctgaactggtatcagcagaagcccgacencoding scFvggcaccgtcaagctgctgatctaccacaccagccggctgcacagcggcgtgcccagccggtttagcggcagcggctccggcaccgactacagcctgaccatctccaacctggaacaggaagatatcgccacctacttttgccagcagggcaacacactgccctacacctttggcggcggaacaaagctggaaatcaccggcagcacctccggcagcggcaagcctggcagcggcgagggcagcaccaagggcgaggtgaagctgcaggaaagcggccctggcctggtggcccccagccagagcctgagcgtgacctgcaccgtgagcggcgtgagcctgcccgactacggcgtgagctggatccggcagccccccaggaagggcctggaatggctgggcgtgatctggggcagcgagaccacctactacaacagcgccctgaagagccggctgaccatcatcaaggacaacagcaagagccaggtgttcctgaagatgaacagcctgcagaccgacgacaccgccatctactactgcgccaagcactactactacggcggcagctacgccatggactactggggccagggcaccagcgtgaccgtgagcagc 26RASQDISKYLN FMC63 CDR L1 27 SRLHSGV FMC63 CDR L2 28 GNTLPYTFGFMC63 CDR L3 29 DYGVS FMC63 CDR H1 30 VIWGSETTYYNSALKS FMC63 CDR H2 31YAMDYWG FMC63 CDR H3 32 EVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRFMC63 VH KGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSS 33DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPD FMC63 VLGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIA TYFCQQGNTLPYTFGGGTKLEIT 34DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPD FMC63 scFvGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGSTSGSGKPGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSS 35 KASQNVGTNVA SJ25C1 CDR L1 36SATYRNS SJ25C1 CDR L2 37 QQYNRYPYT SJ25C1 CDR L3 38 SYWMN SJ25C1 CDR H139 QIYPGDGDTNYNGKFKG SJ25C1 CDR H2 40 KTISSVVDFYFDY SJ25C1 CDR H3 41EVKLQQSGAELVRPGSSVKISCKASGYAFSSYWMNVVVKQR SJ25C1 VHPGQGLEWIGQIYPGDGDTNYNGKFKGQATLTADKSSSTAYMQLSGLTSEDSAVYFCARKTISSVVDFYFDYWGQGTTVTVS S 42DIELTQSPKFMSTSVGDRVSVTCKASQNVGTNVAWYQQKP SJ25C1 VLGQSPKPLIYSATYRNSGVPDRFTGSGSGTDFTLTITNVQSKD LADYFCQQYNRYPYTSGGGTKLEIKR 43GGGGSGGGGSGGGGS Linker 44 EVKLQQSGAELVRPGSSVKISCKASGYAFSSYWMNWVKQRSJ25C1 scFv PGQGLEWIGQIYPGDGDTNYNGKFKGQATLTADKSSSTAYMQLSGLTSEDSAVYFCARKTISSVVDFYFDYWGQGTTVTVSSGGGGSGGGGSGGGGSDIELTQSPKFMSTSVGDRVSVTCKASQNVGTNVAWYQQKPGQSPKPLIYSATYRNSGVPDRFTGSGSGTDFTLTITNVQSKDLADYFCQQYNRYPYTSGGGTKLEI KR 45 HYYYGGSYAMDY FMC63 HC-CDR3 46 HTSRLHS FMC63 LC- CDR2 47 QQGNTLPYT FMC63 LC- CDR3

1-191. (canceled)
 192. A method for assessing cell surface glycans, themethod comprising: (a)(i) incubating a test composition comprising aplurality of cells under conditions to release one or more glycans fromthe surface of cells in the test composition, wherein a samplecomprising one or more cell surface glycans is generated and (a)(ii)determining the presence, absence, identity and/or level of glycanspresent in the sample, thereby assessing the cell surface glycan profileof the sample; or (b) determining the presence, absence, identity and/orlevel of glycans present in a sample, thereby assessing the cell surfaceglycan profile of the sample, wherein the sample comprises one or moreglycans released from the surface of cells present in a test compositioncomprising a plurality of cells after incubation of the test compositionunder conditions to release the one or more glycans.
 193. The method ofclaim 192, wherein cells in the test cell composition comprise wholecells or intact cells.
 194. The method of claim 192, wherein the testcell composition comprises between 1×10⁶ cells and 5×10⁶ cells,inclusive.
 195. The method of claim 192, wherein the test cellcomposition comprises a concentration of between 1×10⁵ cells/mL and1×10⁸ cells/mL, inclusive, between 1×10⁶ cells/mL and 5×10⁷ cells/mL,inclusive, or between 5×10⁶ cells/mL and 2.5×10⁷ cells/mL, inclusive.196. The method of claim 192, wherein the incubation is carried out inthe presence of an N-glycosidase.
 197. The method of claim 196, whereinthe N-glycosidase is a peptide N-glycosidase (PNGase) F.
 198. The methodof claim 197, wherein the PNGase F is recombinant.
 199. The method ofclaim 197, wherein the PNGase F comprises a PGNase F of Flavobacteriummeningosepticum, or a portion or mutant thereof that is enzymaticallyactive.
 200. The method of claim 197, wherein the PNGase F comprises theamino acid sequence set forth in SEQ ID NO: 1 or a portion or mutantthereof that is enzymatically active, or an amino acid sequence thatexhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%. 92%. 93%, 94%, 95%,96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:1 or is aportion thereof that is enzymatically active.
 201. The method of claim197, wherein the PNGase F comprises the amino acid sequence set forth inSEQ ID NO:
 1. 202. The method of claim 197, wherein the amount of PNGaseF is 1 unit to 5000 units, inclusive.
 203. The method of claim 192,wherein the incubating the test composition is for an amount of timethat between or between about 5 minutes and 12 hours, 30 minutes and 6hours or 1 hour and 3 hours, each inclusive.
 204. The method of claim192, wherein the incubating the test composition is at a temperaturebetween 25° C. and 39° C. or between 35° C. and 39° C.
 205. The methodof claim 192, wherein, prior to the determining the presence, absence,identity and/or level of glycans present in a sample, the method furthercomprises labeling glycans from the sample with a detectable label. 206.The method of claim 205, wherein the detectable label is a fluorescentlabel and the fluorescent label is or comprises 2-aminobenzamide (2-AB),2-aminobenzoic acid (2-AA), 2-aminopyridine (PA), 2-Aminoacridone(AMAC), 2-aminonaphthalene trisulfonic acid (ANTS), and1-aminopyrene-3,6,8-trisulfonic acid (APTS),3-(Acetylamino)-6-aminoacridin (AA-Ac), 6-Aminoquinoline (6-AQ),7-Aminomethyl-coumarin (AMC), 2-Amino (6-amido-biotinyl) pyridine (BAP),9-Fluorenylmethoxycarbonyl (FMOC)-hydrazide,1,2-Diamino-4,5-methylenedioxy-benzene (DMB), or o-Phenylenediamine(OPD).
 207. The methods of claim 206, wherein the fluorescent labelcomprises a carbamate tagging group, a quinolone fluorophore, and atertiary amine.
 208. The method of claim 192, wherein, prior todetermining the presence, absence, identity and/or level of the one ormore glycans, the sample is subjected to glycan purification orenrichment.
 209. The method of claim 192, wherein determining thepresence, absence, identity and/or level of the one or more glycanscomprises subjecting the sample to mass spectrometry.
 210. The method ofclaim 192, wherein determining the presence, absence, identity and/orlevel of glycans comprises subjecting the sample to liquidchromatography (LC) followed by mass spectrometry.
 211. The method ofclaim 210, wherein the liquid chromatography is hydrophilic interactionchromatography (HILIC).
 212. The method of claim 209, wherein the massspectrometry comprises ESI-MS or-tandem mass spectrometry (MS/MS). 213.The method of claim 192, wherein the determining the presence, absence,identity and/or level of the one or more glycans comprises analyzing oneor more glycan structure or structures for branching, linkages betweenmonosaccharides and/or location of monosaccharides.
 214. The method ofclaim 192, wherein the one or more glycans comprises high mannoseN-glycans, bisected and Sialyl Lewis^(X) N-glycans, and/or N-acetyllactosamine containing N-glycans.
 215. The method of claim 192, whereinthe cells comprise stem cells, said stem cells comprising immune cells,white blood cells, peripheral blood mononuclear cells (PBMC),lymphocytes, or unfractionated T cells.
 216. The method of claim 192,wherein the cells comprise T cells that are CD4+ and/or CD8+ T cells orthe test cell composition comprises T cells that are CD4+ and/or CD8+ Tcells.
 217. The method of claim 192, wherein the cells express arecombinant receptor or the test composition comprises cells expressinga recombinant receptor.
 218. The method of claim 217, wherein therecombinant receptor is or comprises a chimeric receptor and/or arecombinant antigen receptor.
 219. The method of claim 217, wherein therecombinant receptor is or comprises a T cell receptor or a chimericantigen receptor (CAR).
 220. A method of assaying a cell composition,the method comprising: (a)(i) assessing the cell surface glycan profilein a sample from a test cell composition comprising a plurality of cellsaccording to the method of claim 1 and (a)(ii) comparing the cellsurface glycan profile of the sample to the cell surface glycan profileof a reference sample; or (b) comparing the cell surface glycan profileof a sample compared to the cell surface profile of a reference sample,wherein cell surface glycan profile of the sample is or has beendetermined according to the method of claim 1 from a test cellcomposition comprising a plurality of cells.
 221. A method formanufacturing a cell composition, comprising incubating and/orcontacting an input composition comprising a plurality of cells with oneor more agents and/or under one or more conditions thereby generatingthe cell composition, wherein the cell composition comprises one or aplurality of cells that are genetically, phenotypically, and/orfunctionally different from one or a plurality of cells from the inputcomposition, and wherein the cell composition comprises one or aplurality of cells that comprise a cell surface glycan profilecomprising one or more target glycans and/or each of the one or moretarget glycans in the cell surface glycan profile differs by no morethan 25% from the cell surface glycan profile or each of the one or moretarget glycans to the total glycans present in a reference sample,wherein the cell surface glycan profile comprises glycans released fromthe surface of cells in the cell composition.
 222. The method of claim221, wherein the cell surface glycan profile or the one or more targetglycans is determined according to the method of claim
 1. 223. Themethod of claim 221, wherein the cell composition comprises cellspresent in an apheresis product or a leukapheresis product or cellsderived therefrom.
 224. The method of claim 221, wherein the cellscomprise stem cells, that are immune cells, white blood cells,peripheral blood mononuclear cells (PBMC), lymphocytes, orunfractionated T cells.
 225. The method of claim 221, wherein the cellscomprise T cells that are CD4+ and/or CD8+ T cells.
 226. A method forscreening one or more test agents or conditions on a cell composition,comprising: (a)(i) assessing a cell surface glycan profile in a samplefrom a test cell composition, wherein the test cell composition is or isderived from a source composition that has been incubated or treated inthe presence of one or more test agents or conditions and (a)(ii)comparing the cell surface glycan profile of the sample to the cellsurface glycan profile of a reference sample, the reference samplecomprising one or more target glycans; or (b) comparing the cell surfaceglycan profile of a sample compared to the cell surface glycan profileof a reference sample, wherein the sample is from a test cellcomposition that is or is derived from a source composition that hasbeen incubated or treated in the presence of one or more test agents orconditions.
 227. The method of claim 226, wherein the cell surfaceglycan profile is determined according to the method of claim
 192. 228.A method of detecting a presence, absence, identity, and/or level of oneor more substances in a cell composition, the method comprising: (a)assessing the cell surface glycan profile in a sample from a test cellcomposition comprising a plurality of cells according to the method ofclaim 192, wherein the plurality of cells are from or are derived from acell type; and (b) identifying one or more non-native glycans in thecell surface glycan profile that are not synthesized and/or expressed bycells of the cell type.